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.

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.

Effects of visual impairment on stroke parameters in Paralympic swimmers.

PURPOSE: To examine the relationship between degree of vision and stroking parameters in male and female Paralympic swimmers with visual impairment during the 50- and 100-m freestyle events. METHODS: A video analysis was conducted at the 1996 Paralympic Games in which swimmers competed in three groups based on degree of impairment (S11, S12, and S13; S11 least amount of vision). A video camera placed 25 m from the start, perpendicular to the swimming direction, recorded the performance of each swimmer during the clean swim phase. Variables measured included total race time, clean swimming speed (CSS), stroke rate (SR), stroke length (SL), and stroke index (SI = CSS x SL). Comparisons of performance were made between the classes and between men and women. RESULTS: The men showed no significant differences between S12 and S13 on any of the variables or between all three classes on SL and SI. The S11 swimmers demonstrated a significantly slower total race time and CSS in both events. In the women, an increase in class was associated with a decrease in total race time, faster CSS, and increase in SI. In comparing men and women, men demonstrated a significantly faster CSS and total race time during both events, whereas no differences were observed in SR. CONCLUSION: Stroke parameters during the clean swim phase were affected by visual impairment in both men and women. The male classes, however, were not clearly distinct from each other based on the swimming variables measured, as no significant differences were found between S12 and S13 in either event. With the exception of stroke rate and length, performance of the women tended to increase with an increase in class.

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.

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.

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.

The effects of surface anaesthesia on the autonomic dysreflexia response during functional electrical stimulation.

Recently, increases in blood pressure (BP) and concomitant bradycardia, suggestive of autonomic dysreflexia (AD), have been documented during functional electrical stimulation (FES) in individuals with a high spinal cord injury (SCI). If uncontrolled, this response could preclude the safe use of FES among such individuals. FES induced pain is partly related to stimulation of skin nociceptors. Therefore, measures to reduce skin sensitivity may reduce the risk of AD during FES. The purpose of this study was to determine if topical anaesthetic applied over the site of electrical stimulation could minimize the AD cardiovascular and hormonal responses to FES in individuals with SCI above the T6 level. Seven subjects with a SCI above T6 received FES to the quadriceps muscle of each leg under two conditions on two different testing days. The two treatment conditions, topical anaesthetic and placebo creams, were double blinded and randomized. The cream was administered to an area the size of the electrode (10 x 10 cm) 1 h prior to stimulation. Stimulation began at 0 mAmps and increased by 16 mAmps every 2 min until an intensity of 160 mAmps was achieved. HR and BP were measured at each stimulation intensity level. Catecholamines were analyzed three times during the stimulation protocol (pre, mid and post stimulation intensities). At the end of the stimulation protocol, FES induced isometric quadriceps contraction force at 160 mAmps intensity was measured using a hand held dynamometer. As FES stimulation intensity increased, significant rises in systolic and diastolic BP were seen, with a concomitant progressive drop in HR. The AD response to stimulation was not significantly different between the topical anaesthetic and placebo conditions. Serum catecholamine (epinephrine and norepinephrine) levels tended to rise with increasing FES intensity levels but did not reach statistical significance. The two treatment conditions did not significantly affect serum catecholamine levels or FES-induced quadriceps contraction force. In summary, FES application to the quadriceps muscle in high level SCI subjects resulted in significant increases in BP, decreases in HR (AD-like response), a trend towards elevations in catecholamine levels, and no difference in quadriceps muscular strength. However, these responses were unaffected by the use of topical anaesthetic cream on the skin at the stimulation site. This suggests that other mechanisms than skin nociception are operative in FES-induced AD.

Use of nucleotide sequence data to identify a microsporidian pathogen of Pieris rapae (Lepidoptera, Pieridae).

Nucleotide sequence was determined for a portion of genomic DNA which spans the V4 variable region of the small subunit ribosomal RNA gene of an unidentified microsporidium from the cabbage white butterfly, Pieris rapae (174 base pairs). Comparison with equivalent sequence data obtained here for two other microsporidian species, Nosema bombycis (240 base pairs) and Nosema bombi (200 base pairs), and from the GenBank database for 11 other microsporidian species suggests that the unidentified species from P. rapae is most closely related to some Vairimorpha species. Light and electron microscopic observations of the developmental stages of this parasite were in accord with this. Infection experiments conducted at 20 and 26 degrees C demonstrated temperature-dependent dimorphism, with the production of both binucleate free spores (mean dimensions: 3.8 x 1.8 microns; 10-13 polar filament coils) and membrane-bound uninucleate octospores (mean dimensions: 3.1 x 1.9 microns). Macrospores (mean dimensions 8.0 x 2.1 microns) were also observed. Sites of infection were the gut epithelium, the Malpighian tubules, the salivary glands, and the fat body. Infections were found in all insect life stages, including the egg. This microsporidium was found to be indistinguishable from both Nosema mesnili (Paillot) and Microsporidium (Thelohania) mesnili (Paillot) and we propose that these species be combined and transferred to the genus Vairimorpha Pilley.

DNA probes for two Microsporidia, Nosema bombycis and Nosema costelytrae.

Two DNA fragments which hybridize specifically with DNA of Nosema bombycis and Nosema costelytrae, respectively, were obtained from genomic DNA of each microsporidian species and sequenced. Neither fragment hybridized with genomic DNA from four other microsporidian isolates tested: Nosema apis, Vairimorpha sp. from cabbage white butterfly (Pieris rapae), and two isolates of Vavraia oncoperae, one from New Zealand grass grubs, Costelytrae zealandica, and another from porina caterpillars, Wiseana spp. The probe for N. bombycis did not hybridize with genomic DNA from N. costelytrae or with DNA from silkworms (Bombyx mori), the primary insect host of this microsporidium. Likewise, the probe for N. costelytrae did not hybridize with genomic DNA from N. bombycis or with DNA from grass grubs (C. zealandica). Both fragments were AT-rich (59 and 79% of total bases, respectively), had G+C/A+T ratios of 0.70 and 0.25, respectively, and represented repeated sequences dispersed throughout the genome.

Dynamic performance assessment of selected sport shoes on impact forces.

Few investigators have evaluated the performance characteristics of non-running sport shoes. The purpose of this study was to assess the dynamic performance characteristics of four different shoe models during landings. Five male subjects performed 25 voluntary hanging drop landings (60 cm) onto a force platform (1000 Hz) for each of four shoe conditions (C1 and C2 = basketball shoe, C3 = running shoe, C4 = volleyball shoe). Ground reaction force data were evaluated for maximum forefoot (F1) and rearfoot (F2) impact forces as well as the respective times of occurrence of these events (T1, T2). Results of the group data analysis indicated a preferential performance rank order of C1, C3, C4, C2 although significant interaction effects were observed, indicating a need for single-subject analyses. Three techniques were incorporated to assess individual subject condition differences, all of which elicited unique rank orders for the shoes although each identified C1 as the "best" shoe condition. The results of the study support the necessity for within-subject analyses conducted with an adequate number of trials when attempting to detect subtle performance differences that may exist between various sport shoes. Whether the observed statistically significant differences are biomechanically meaningful remains an important unanswered question.

Osteoid osteoma: excision with scintimetric guidance.

The definite diagnosis of osteoid osteoma relies on the demonstration of the nidus, best shown by CT, which also provides precise preoperative localization of the nidus. While bone-block excision to remove the nidus is feasible in the long bones, there may be unacceptable sequelae in the vertebral column and small bones of the hands. By precisely localizing the nidus, radionuclide scintimetry permits excellent therapeutic results, with minimal morbidity.

Kinetics of technetium 99m labelled macroaggregated albumin in humans.

The uptake and elimination of 99Tcm labelled MAA were followed by gamma camera and computer over a period of 36 hours in patients undergoing lung scanning and venography. Lungs, stomach, kidneys, GI tract, bladder and thyroid showed significant concentrations of activity at various times after the injection of radiopharmaceutical, with carefully controlled labelling efficiency. There was no indication of accumulation in the liver or spleen. Activity versus time curves were constructed. The lung curve had approximately bi-exponential form with components of effective half-lives 0.88 and 4.56 h. Areas beneath the curves gave cumulated activities for each organ and, using S values (absorbed dose per unit cumulated activity) from MIRD tables, absorbed doses from self-irradiation were calculated for each organ.