Tri-trophic effects of transgenic insect-resistant tobacco expressing a protease inhibitor or a biotin-binding protein on adults of the predatory carabid beetle Ctenognathus novaezelandiae.

Tri-trophic impacts on adult predatory carabid beetles, Ctenognathus novaezelandiae, of insect-resistant transgenic tobacco plants expressing a serine protease inhibitor, bovine spleen trypsin inhibitor (BSTI), or a biotin-binding protein, avidin, were investigated. Both proteins could potentially affect this beetle, since avidin is known to be insecticidal to many beetle species and C. novaezelandiae midguts were shown to contain high levels of trypsin, a protease powerfully inhibited by bovine pancreatic trypsin inhibitor (a BSTI homologue) in vitro. Newly emerged field-collected adult C. novaezelandiae were fed exclusively for 280 days on Spodoptera litura larvae raised either on non-transgenic control, transgenic avidin (55 ppm) or transgenic BSTI (68 ppm) tobacco. Despite this long-term exclusive diet, there was no treatment effect on survival or fecundity and only minor and transient effects on beetles were observed. Data pooled across time and genders showed control-prey-fed beetles weighed 3% more than BSTI-prey-fed beetles and avidin-prey-fed beetles consumed 3-4% fewer prey than control- or BSTI-prey-fed individuals. Females in all treatments gained more mass and survived longer than males. Low exposure to the proteins because of dilution and deactivation within the prey is the most likely explanation for the lack of tri-trophic effects observed. Aditionally, the presence of a digestive chymotrypsin only partially inhibited by BSTI may provide an alternative path for proteolysis.

Biosafety testing of genetically modified ryegrass (Lolium perenne) using a model for the optimum selection of test invertebrates.

Selection of test species for use in biosafety evaluation of genetically modified plants is challenging but important, as regulators in many jurisdictions require tests to determine the potential for adverse environmental impacts before the release of plants into the environment. This contribution provides an example of an evidence-based process whereby species from the receiving environment can be ranked in order of susceptibility to potential impact, and guide test species selection. The case study used for this example was ryegrass, a forage plant, which had been modified to produce elevated levels of the lipid triacylglyceride. The previously described priority ranking of nontarget invertebrates model (PRONTI), designed to rank invertebrates for biosafety testing, has been adapted for use with these plants, which could, potentially, be beneficial to invertebrate populations, and applied to data on 246 known pasture invertebrate species. The output from the model for the top 20 ranked pasture invertebrate species is discussed, the attributes of these are considered along with the level of uncertainty in the information used. Consideration is given to how the model output can be interpreted and used in a biosafety risk assessment. While some subjectivity is involved in establishing the scores, all invertebrate species are subjected to the same analysis, and treated equally. In this way, regulators have a method of a risk assessment that is evidence-based, and transparent in its assumptions thereby avoiding potential for bias.

No impact of transgenic nptII-leafy Pinus radiata (Pinales: Pinaceae) on Pseudocoremia suavis (Lepidoptera: Geometridae) or its endoparasitoid Meteorus pulchricornis (Hymenoptera: Braconidae).

To investigate the biosafety to insects of transgenic Pinus radiata D. Don containing the antibiotic resistance marker gene nptII and the reproductive control gene leafy, bioassays were conducted with an endemic lepidopteran pest of New Zealand plantation pine forests and a hymenopteran endoparasitoid. Larvae of the common forest looper, Pseudocoremia suavis (Butler), were fed from hatching on P. radiata needles from either one of two nptII-leafy transgenic clones, or an isogenic unmodified control line. For both unparasitized P. suavis and those parasitized by Meteorus pulchricornis (Wesmael), consuming transgenic versus control pine had no impact on larval growth rate or mass at any age, larval duration, survival, pupation or successful emergence as an adult. Total larval duration was 1 d (3%) longer in larvae fed nptII-2 than nptII-1, but this difference was considered trivial and neither differed from the control. In unparasitized P. suavis larvae, pine type consumed did not affect rate of pupation or adult emergence, pupal mass, or pupal duration. Pine type had no effect on the duration or survival of M. pulchricornis larval or pupal stages, mass of cocoons, stage at which they died, adult emergence, or fecundity. Parasitism by M. pulchricornis reduced P. suavis larval growth rate, increased the duration of the third larval stadium, and resulted in the death of all host larvae before pupation. The lack of impact of an exclusive diet of nptII-leafy transgenic pines on the life history of P. suavis and M. pulchricornis suggests that transgenic plantation pines expressing nptII are unlikely to affect insect populations in the field.

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.

Ultrastructural changes to the midgut of the black field cricket (Teleogryllus commodus) following ingestion of potato protease inhibitor II.

Ultrastructural changes to the midgut epithelium of nymphs of the black field cricket (Teleogryllus commodus) after ingestion of potato protease inhibitor II (PPI-II) (0.6% (w/v) in artificial diet) were determined by light and electron microscopy. Crickets fed diet containing PPI-II grew more slowly than those fed control diet and changes observed to the PPI-II-fed nymphs included reduction of midgut wall depth, vacuolisation of the epithelial cells, swelling of the microvilli, cellular protrusions into the midgut and eventual rupture of individual or small groups of epithelial cells. These changes were first seen 2 days after PPI-II ingestion. Complete disintegration of the midgut to the basement membrane was not seen during the 27-day observation period and repair and regeneration of pockets of epithelial cells was observed. Immunocytochemistry revealed that PPI-II was localised within the ectoperitrophic matrix space of the gut. The location of the peritrophic matrix was determined by labelling with wheat germ agglutinin (WGA), but no rupture of this structure was observed in PPI-II-fed nymphs.

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.