Responses of 9 lepidopteran species to Bacillus thuringiensis: How useful is phylogenetic relatedness for selecting surrogate species for nontarget arthropod risk assessment?

To evaluate phylogenetic relatedness as a proxy for susceptibility to Bacillus thuringiensis (Bt) when selecting species to act as surrogates for others in prerelease testing, we examined the responses of 11 laboratory-reared lepidopteran colonies, comprising members of 2 families, 5 genera, and 9 species, to a commercial Bt preparation. Survival, pupal mass, and timing of pupation and adult emergence of 2 noctuids (Spodoptera litura and Helicoverpa armigera) and 7 tortricids (Cnephasia jactatana, Ctenopseustis obliquana, Ctenopseustis herana,Planotortrix octo, Planotortrix notophaea,Planotortrix excessana [2 different laboratory colonies], and Epiphyas postvittana [2 colonies]) were examined after feeding first instar larvae with artificial diet containing 5 µL/100 mL Dipel ES (Bt subsp. kurstaki). Bt caused significant larval mortality in all species except S. litura, in which only pupation was delayed compared with untreated controls. Neither of the noctuid species tested would act as a suitable surrogate for the other in tests of Bt impacts on survival. With the exception of the 2 colonies of E. postvittana, which differed from each other not only in their responses to Bt but also in their development times when not treated with Bt, species within each tortricid genus had similar responses to Bt and thus could act as surrogates for each other. Members of different genera within this family could represent each other only if relatively coarse measurement endpoints (e.g., toxic or not) were considered adequate for assessing risks to nontarget species in the field.

Triterpene acids from apple peel inhibit lepidopteran larval midgut lipases and larval growth.

Fruit extracts from apple, kiwifruit, feijoa, boysenberry, and blueberry were screened for the presence of lipase inhibitory compounds against lepidopteran larval midgut crude extracts. From 120 extracts, six showed significant inhibition with an extract from the peel of Malus × domestica cv. "Big Red" showing highest levels of inhibition. Because this sample was the only apple peel sample in the initial screen, a survey of peels from seven apple cultivars was undertaken and showed that, despite considerable variation, all had inhibitory activity. Successive solvent fractionation and LC-MS of cv. "Big Red" apple peel extract identified triterpene acids as the most important inhibitory compounds, of which ursolic acid and oleanolic acid were the major components and oxo- and hydroxyl-triterpene acids were minor components. When ursolic acid was incorporated into artificial diet and fed to Epiphyas postvittana Walker (Tortricidae: Lepidoptera) larvae at 0.16% w/v, a significant decrease in larval weight was observed after 21 days. This concentration of ursolic acid is less than half the concentration reported in the skin of some apple cultivars.

The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana (Walker) (Tortricidae, Lepidoptera).

The effects of the lipase inhibitor, tetrahydrolipstatin (THL), on neonate Epiphyas postvittana (Walker) (Lepidoptera, Tortricidae) larvae were investigated by feeding on control artificial diets (with and without 2% ethanol) and diets containing 2% ethanol and one of three concentrations of THL (0.011%, 0.037% and 0.11%). Small but significant reductions in growth rate, percent pupation and time to pupation were observed for larvae feeding on 2% ethanol control diet compared with standard control diet, but larger reductions in all parameters occurred with increasing THL concentration. Third instar larvae fed 0.011% THL in the diet had 40% of the midgut lipase activity in the relevant control larvae and showed up-regulation of gene expression of the gastric lipase-like family but not the pancreatic lipase-like family of midgut lipases.

The use of biotin-binding proteins for insect control.

Biotin-binding proteins (BBPs), expressed in transgenic plants, are insecticidal to a very wide range of insects. The expression levels required are generally low (approximately 100 ppm), and although higher than required for Bacillus thuringiensis (Bt) delta-endotoxins, BBPs are effective across a broader range of insect orders and other invertebrates than the Bt Cry proteins. Avidin and streptavidin, in particular, have been reported as causing death or severe growth reduction in at least 40 species of insects across five insect orders (Lepidoptera, Coleoptera, Orthoptera, Diptera, and leaf-eating Hymenoptera) and mites. In addition, due largely to its rapid dilution in ecosystems, no adverse impacts on nontarget microorganisms or invertebrates have been recorded. Because the target, biotin, cannot itself be modified to prevent it binding to BBPs and remain effective as a vitamin, the major avenue open to insects to develop resistance is unavailable. Two properties of the biotin-avidin complex make it highly suitable for use in transgenic plant crop protection strategies against a large range of insects; its extreme stability and its resistance to proteolysis. However, because the nutritional value of the plant could potentially be compromised in the absence of biotin supplementation, its use in nonfood crops such as fiber, forestry, and biofuel crops is seen as the most suitable initial focus for this technology.

Expression of various biotin-binding proteins in transgenic tobacco confers resistance to potato tuber moth, Phthorimaea operculella (Zeller) (fam. Gelechiidae).

The high affinity biotin-binding proteins (BBPs) avidin and streptavidin are established insecticidal agents, effective against a range of insect pests. Earlier work showed that, when expressed in planta, full length avidin and a truncated form of streptavidin are highly insecticidal. More recently, a wide range of BBPs, found in diverse organisms or engineered for various biotechnological applications have been reported. However, their effectiveness as plant-based insecticides has not been established. Here we report in planta expression of three different genes, designed to produce BBP variant proteins in the vacuole. The first was mature full length chicken avidin, the second a circularly permuted dual chain chicken avidin, and the third was an avidin homologue, a native bradavidin from Bradyrhyzobium japonicum. All three proteins were expressed in Nicotiana tabacum (tobacco). The transgenic tobacco lines were healthy, phenotypically normal and, when subjected to bioassay, resistant to the important cosmopolitan pest, potato tuber moth (Phthorimaea operculella) larvae at concentrations of ~50 ppm.

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods.

The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material.

Transgenic tobacco and apple plants expressing biotin-binding proteins are resistant to two cosmopolitan insect pests, potato tuber moth and lightbrown apple moth, respectively.

Tobacco (Nicotiana tabacum cv. Samsun) and apple (Malus x domestica cv. Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation. ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple. Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam. Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam. Tortricidae). More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation. Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days. Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.

The expression of a mammalian proteinase inhibitor, bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae.

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3' and 5' changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.