Appl. Entomol. Zool. 40 (1): 151–159 (2005) http://odokon.ac.affrc.go.jp/
Effects of transgenic Bt corn pollen on a non-target lycaenid butterfly, Pseudozizeeria maha
Yoichi SHIRAI* and Mami TAKAHASHI National Institute for Agro-Environmental Sciences; Tsukuba, Ibaraki 305–0856, Japan (Received 22 April 2004; Accepted 2 November 2004)
Abstract To evaluate the effect of pollen released from transgenic insecticidal corn on non-target lepidopteran insects, corn pollen deposition density on the leaves of sunflower and black nightshade was measured near a cornfield. At 12 d from the start of anthesis, the highest cumulative pollen density on leaves was approximately 160 grains per cm2 at 1 m from the edge of the cornfield, falling to 20 grains at 5 m and less than 10 grains at 10 m. The pollen density calculated using a mathematical model in a previous study evidently had overestimated values. To evaluate precisely the effect of corn pollen expressing Bacillus thuringiensis (Bt) endotoxin (Cry1Ab) on the survival of lepidopteran larvae, we im- proved the bioassay methods using the pale grass blue, Pseudozizeeria maha, the leaf disc of the wood sorrel, Oxalis corniculata, and transgenic Bt corn (Event-176). When the surface of the leaf was pretreated with a small amount of 80% acetone solution, the preselected pollen dose was successfully applied onto the leaf disc. Larval survival of P. maha was significantly affected at pollen density of more than 20 grains per cm2 on the leaf disc. It is unlikely that pollens from Bt corn expressing Cry1Ab have wide-scaled deleterious effects on non-target P. maha near cornfields, because of low pollen deposition dose on the leaves.
Key words: Bt; Cry1Ab; non-target insect; pollen density; Pseudozizeeria maha
Schmitz et al., 2003) and the Netherlands (Knols INTRODUCTION and Dicke, 2003), there are still precautions against There is much concern about insecticidal trans- the risk of Bt-corn pollen to non-target lepidopter- genic crops regarding the potential risks to non-tar- ans. In addition, Losey et al. (2003) imply that get arthropods and ecosystems (Cannon, 2000; there is negative impact on non-target lepidopteran Conner et al., 2003; Knols and Dicke, 2003). species including moths other than the monarch Losey et al. (1999) have suggested that pollen re- butterfly. leased from transgenic corn expressing Bacillus In Japan, a bioassay using the pale grass blue, thuringiensis (Bt) endotoxin may harm the larvae Pseudozizeeria maha was appended to the risk as- of a non-target lepidopteran, the monarch butterfly, sessment for environments associated with trans- Danaus plexippus. Many subsequent studies have genic Bt-corn, according to the preliminary studies shown that such a risk to this butterfly rarely occurs (Matsuo et al., 2002). This bioassay method, how- in the field because small numbers of corn pollen ever, is laborious and does not allow the accurate are deposited on the leaves of the milkweed, Ascle- evaluation of corn pollen dose. In addition, Matsuo pias syriaca, the host plant of the monarch butter- et al. (2002) have estimated corn pollen density fly within and near cornfields (Pleasants et al., using a mathematical model instead of the actual 2001; Sears et al., 2001; Jesse and Obrycki, 2003). pollen dose deposited on leaves near the cornfield. Currently, each government authority imposes risk In February 2004, the Cartagena Protocol on assessment on non-target arthropods through na- biosafety related to the regulation and management tional regulation prior to the approval of commer- of transgenic crop cultivation came into force in cial field cultivation of insecticidal transgenic crops Japan, and the procedures for risk assessment of (Nap et al., 2003). In Germany (Felke et al., 2002; environment at the petition for field cultivation
*To whom correspondence should be addressed at: E-mail: fl[email protected] DOI: 10.1303/aez.2005.151
151 152 Y. SHIRAI and M. TAKAHASHI were revised (http://www.biodic.go.jp/cbd/biosafety/ each leaf surface with a cork borer (1.6-cm diame- index.html). It is necessary to establish sound ter) and the number of pollen grains was counted guidelines for risk assessment and bioassay meth- within the viewing area (ca. 2 cm2) under a stere- ods corresponding to actual field conditions. In the omicroscope. To measure the ambient pollen dose, present study, we measured corn pollen density on from August 30 to September 14, each micro slide the leaves of plants on which herbivorous insects glass (18 18 mm) was set at 1.3 m above ground feed near a cornfield to improve bioassay methods level on the pollen sampler (Durham type) in four for P. maha larvae using transgenic Bt corn (Event- directions at 0 m (the edge of cornfield). The slide 176). glass coated with Vaseline was replaced everyday, and the number of pollen grains on the slide glass was counted under a stereomicroscope. MATERIALS AND METHODS Collection of Bt corn pollen and evaluation of Pollen deposition on leaves near cornfields. endotoxin. Transgenic corn expressing Bt endo- Non-transgenic dent corn (snow-dent) was planted toxin (Cry1Ab), Event-176 (var. Max.21) was in the plots (7 7 m) on the farm of the National planted in a segregated-experiment field of NIAES Institute for Agro-Environmental Sciences on June 20, 2003. Before anthesis, all tassels were (NIAES), Tsukuba, in June 19, 2003. Corn rows bagged, and pollen was collected between August and plants were 0.7 m and 0.2 m wide, respectively. 18 and 29. The pollen was sieved through a screen At early anthesis, on August 30, each of five potted mesh to remove materials other than pollen and sunflower plants, Helianthus annuus (ca. 60 cm was then kept in a sealed plastic box at 30°C. stem height) were placed in four directions at 0, 1, Non-transgenic corn (N4640) was planted in 2002, 2, 5 and 10 m from the edge of the cornfield. Simi- and the corn pollen stored at 30°C was used for larly, five potted sunflower plants were placed in a the present study. For Event-176 and N4640, the NE-direction at 20 and 30 m from the edge. For the expression level of Bt endotoxin (Cry1Ab) in black nightshade, Solanum nigrum, each of two pollen was determined by enzyme-linked im- potted plants (ca. 50 cm stem height) were munosorbent assay (ELISA) using a GMO Check- arranged in four directions at 0, 1, 2 and 5 m from Bt1 Maize Test Kit (Strategic Diagnostics Inc.). the edge (Fig. 1). One leaf was gently removed The method described by the manufacturer is for from the upper part of each potted plant every 2 d corn grain material. We adapted it for corn pollen from September 2 to 11, and the leaves were samples as follows. The pollen was suspended in placed in a 9-cm petri dish with moist filter paper. 10 mM phosphate buffer (pH 7.4) containing In the laboratory, a slight indentation was made on 2.7 mM KCl, 13.7 mM NaCl, 100 mM phenylmethyl-
Fig. 1. Locations of cornfield and potted sunflower plants ( ). Effect of Bt Corn Pollen on Non-Target Butterfly 153 sulfonyl fluoride, 1 mg/ml leupeptine, and 0.1% 5°C. If pollen doses of 10 grains and 1,000 grains Triton X, and the suspension was sonicated using are applied to the leaf disc, 0.184 mg and 18.4 mg, an Astrason Ultrasonic Processor XL 2020 (Heat respectively, of pollen is weighed and added to 1 ml Systems Inc.). The debris was removed by centrifu- of distilled water. At bioassay, after churning, we gation at 10,000 rpm for 5 min. To quantitatively pipetted 10 ml of the pollen suspension onto each evaluate an expression level of Bt endotoxin, the leaf disc. During the experiment described below, authentic sample of Cry1Ab endotoxin (0.147 mg the number and weight of pollen grains was mea- per ml) was used as a control for ELISA. sured every 7 d, and each time, a pollen suspension Insect and plant leaves. In and near cornfields was prepared on the basis of the grain-weight ratio in Japan, there are no representative non-target lep- because pollen weight fluctuates depending on idopteran species corresponding to the monarch storage conditions. butterfly of North America. Matsuo et al. (2002) Preliminary bioassay. The surface of O. cornic- showed that larvae of pale grass blue, P. maha, ulata leaf is hydrophobic, however, the leaf does were most sensitive to Bt endotoxin (Cry1Ab) not repel water when the surface is treated with a among four species of butterfly (P. maha, Pieris small amount (ca. 2 or 3 ml) of 80% acetone solu- rapae, Lampides boeticus and Papilio xuthus). We tion, and it allows the pollen suspension to be ap- selected P. maha as the test insect for assessing risk plied uniformly onto a leaf disc. From a prelimi- of Bt corn pollen to non-target species, because P. nary study, it was confirmed that the leaf of O. cor- maha is frequently found near cornfields and is rel- niculata is burned by the application of 100% ace- atively easily reared in the laboratory. P. maha uses tone, whereas it repels water with less than a 70% O. corniculata leaf as a host plant, however, we acetone solution. To evaluate the effect of pretreat- could not count the number of corn pollen grains ment with 80% acetone solution and the validity of deposited on O. corniculata leaves as in the case of this bioassay procedure, first instar larvae of P. the sunflower and black nightshade leaves de- maha were reared on (a) an intact O. corniculata scribed above because the leaves of O. corniculata leaf disc, (b) a leaf disc treated with 80% acetone close in the night. The sunflower and black night- solution, and (c) a leaf disc to which 10 ml of shade leaves were used as a substitute for O. cor- pollen suspension containing the transgenic Bt niculata, although there are no butterfly species pollen dose of 1,000 grains per cm2 had been ap- which use these two plants as host plant in Japan. plied after pretreatment with 80% acetone solution. Preparation of the corn pollen suspension. Besides these three treatments, 18.4 mg of pollen The number of pollen grains and their weight were was added directly to 1 ml of 80% acetone solu- measured to create a suspension containing the tion, and larvae were reared on a leaf disc treated preselected pollen dose. From a frozen box, a small with 10 ml of this suspension (a pollen dose of amount of corn pollen was applied to a micro cover 1,000 grains per cm2) (d). For two treatments (c glass (18 18 mm) coated with Vaseline and was and d), the corn pollen of Event-176 was used and weighed using a micro balance (MX-5, Mettler the surface of the leaf disc was dried for 2 h at Toledo). The number of grains on the cover glass 25°C and 60% RH. was counted under a stereomicroscope. This mea- Three pairs of wild P. maha adults were intro- surement was replicated ten times. From this rela- duced into a mesh cage (50 30 35 cm high) with tionship between the number and weight of pollen a potted wood sorrel, Oxalis corniculata. The grains, a suspension of pollen (10 ml) was prepared hatched larvae from eggs on O. corniculata leaves to create the preselected pollen dose on a leaf disc were used for the bioassay. Each treated leaf disc of O. corniculata (1-cm diameter, 0.785 cm2). was placed in a plastic sealed dish (9-cm diameter, Thus, when 1,000 grains of pollen weighs 4.5-cm depth) with moist filter paper, and four first 0.234 mg, 0.01837 mg of pollen is required in a instar larvae of P. maha (2 d after hatching) were 10 ml of suspension in order to create the leaf disc introduced onto the leaf disc. The leaf disc was re- (1 cm diameter) at a pollen dose of 100 grains per placed daily, and larval survival was recorded for cm2: 0.01837 100 0.785 0.234 1,000. A total 6d under the constant conditions of 25°C and of 1.84 mg of pollen was added to 1 ml of distilled 16L8D. water in a vial, and this suspension was kept at Effect of Bt corn pollen on larval survival. 154 Y. SHIRAI and M. TAKAHASHI
Using the pollen of Event-176, larval survival of P. maha was evaluated at a pollen dose of 10, 20, 40, 60, 80 and 100 grains per cm2. For each pollen dose, we prepared 1 ml of a suspension of pollen containing the preselected pollen dose by the treat- ment (c) described above, and we pipetted 10 ml of the suspension onto each leaf disc. For the pollen of non-transgenic corn (N4640), as a control, the larval survival of P. maha was evaluated at a pollen dose of 20, 60 and 100 grains per cm2. In both studies, each treated leaf disc was placed in a plas- tic sealed dish (9-cm diameter, 4.5-cm depth) with ns (NE, SE, SW and NW). moist filter paper, and four first instar larvae of P. maha (2 d after hatching) were introduced onto the leaf disc. The leaf discs were replaced daily, and the larval survival was recorded for 6 d under the constant conditions of 25°C and 16L8D. Statistical analysis. Larval survival rates and proportion of larvae molting to the second instar were transformed to arcsin and analyzed by Tukey’s test (StatSoft, 1999).
RESULTS Pollen deposition on leaves near cornfield Corn pollen on slide glass was deposited most densely between Sep. 2 and 6, and then decreased rapidly on and after Sep. 10. For the duration of the study, it rained little and there was a light wind (Table 1). On the sunflower leaves (Table 2), much pollen was counted on Sep. 8 (9 d from the start of anthesis) and Sep. 11 (12 d), and the highest cumu- lative pollen density was 155 grains per cm2 at 1 m
from the edge of the cornfield in a SW-direction on 234567891011121314 Sep. 11. More than 100 grains of pollen were also recorded at four points at 1 m from the edge on
Sep. 8 and Sep. 11. In general, pollen was more in 2003 data during the field experiment Corn pollen deposition on slide glass and meteorological
abundantly deposited at 1m from the edge than at .
0m (field edge), and was limited to less than e1 20 grains at 5 m, and less than 10 grains at 10 m bl Ta from the edge. From Sep. 2 to Sep. 8, the pollen density was less on the leaves in a SE-direction than in the other three directions, but there was no Aug. 30 31 Sep. 1 a
clear relationship with wind direction because no ) dominant wind from a SE-direction was recorded 2 over this period (Table 1). On the black nightshade leaves (Table 3), a lot of pollen was counted on on slide glass (grains/cm wind direction (m/s) ollen densityollen 85.5 60.5 80.0 251.5 565.8 238.8 330.8 624.0 52.8 94.8 169.8 27.5 9.5 5.8 3.5 2.3 Mean values of pollen density on slide glass of pollen samplers which were placed at 0 were of pollen density on slide glass samplers which Mean values m from the cornfield edge in four directio P Sep. 8 (9 d from the start of anthesis), and the high- Rainfall (mm)The most frequent 0.0 ENEMean wind speed 0.5 ENE 0.8 ENE 2.0 0.8 E 0.0 0.9 NE 0.0 0.7 ENE 1.0 E 0.9 0.0 1.1 SW 0.0 1.2 ENE 0.0 NE 0.9 0.0 SSW 1.5 SSW 0.0 0.8 SSE 0.0 ENE 1.3 0.0 SSW 2.4 WSW 0.0 1.1 0.0 1.2 0.0 1.6 1.4 a est cumulative pollen density was 150 grains per cm2 at 1 m from the field edge in a SW-direction. Effect of Bt Corn Pollen on Non-Target Butterfly 155 0.2 1.1 0.2 0.8 0.5 0.1 0.0 1.4 0.0 0.1 0.6 0.4 a ) near a cornfield 1.7 — — 4.8 — — 0.4 0.3 0.7 — — 2.4 — — 0.9 — — 0.5 0.0 1.0 — — 0.7 — — 0.8 — — 0.1 0.0 0.4 — — 0.7 — — 0.2 — — 0.1 0.4 0.9 — — Helianthus annuus 8.2 3.3 0.0 7.4 4.7 0.3 10.4 0.8 18.0 5.2 3.1 1.0 3.0 0.7 7.7 2.2 3.6 0.7 1.5 0.5 1.3 0.0 11.0 0.2 4.0 0.7 0.5 0.1 0.2 0.0 2.9 1.5 Distance from the field edge 40.5 16.3 6.1 0.0 27.9 5.2 30.4 12.2 20.1 19.6 11.2 2.1 23.2 7.1 25.6 11.9 23.6 8.0 0.4 0.8 1.9 1.0 7.8 9.3 15.8 6.2 0.6 0.5 2.4 0.3 26.8 13.7 ) on the leaves of sunflower ( of sunflower ) on the leaves 2 66.9 63.3 46.1 15.6 36.8 33.5 76.3 49.5 37.5 84.1 9.0 6.3 25.6 60.3 16.9 44.1 13.0 42.3 0.9 0.6 28.4 3.8 25.3 34.8 12.2 25.2 0.8 1.2 9.0 3.4 50.4 60.1 Corn pollen density (grains/cm . e2 32.5 155.4 21.2 101.5 36.0 136.5 36.4 66.0 8.5 106.9 4.9 11.7 53.9 112.9 28.6 47.1 30.2 31.2 13.4 3.8 34.7 42.5 30.1 83.8 32.4 55.6 5.2 2.8 41.0 12.4 19.0 94.4 bl 0m 1m 2m 5m 10m 20m 30m Ta b 5). n SW 34.4 SE 38.0 NE 81.7 NW 41.8 SW 18.5 SE 4.7 NE 70.6 NW 57.9 SW 61.7 SE 20.1 NE 37.4 NW 49.3 SW 30.9 SE 10.8 NE 43.5 Direction NW 17.0 SD ( Sep. 11 (at 12 d from start of anthesis) Sep. 8 (at 9 d from start of anthesis) Sep. 5 (at 6 d from start of anthesis) Collection date Sep. 2 (at 3 d from start of anthesis) See Fig. 1 for direction. See Fig. Mean a b 156 Y. SHIRAI and M. TAKAHASHI
Table3. Corn pollen density (grains/cm2) on the leaves of black nightshade (Solanum nigrum) near a cornfield a
Distance from field edge Collection date b Direction 0m 1m 2m 5m
Sep. 2 (at 3 d from start of anthesis) NE 30.8 17.8 19.2 3.1 18.2 8.6 0.6 0.2 SE 3.5 2.2 4.5 0.5 2.2 1.6 0.2 0.2 SW 13.0 5.0 72.6 0.8 31.9 20.2 6.9 1.1 NW 44.8 14.5 53.1 2.5 80.8 26.5 9.8 6.4 Sep. 5 (at 6 d from start of anthesis) NE 8.3 2.0 31.4 11.7 2.6 3.4 0.1 0.2 SE 3.3 0.6 6.1 2.0 0.7 0.9 0.0 0.0 SW 30.9 30.7 87.2 13.4 25.0 1.1 8.4 6.9 NW 48.1 16.4 39.0 1.6 51.5 24.3 7.2 1.7 Sep. 8 (at 9 d from start of anthesis) NE 50.2 17.4 53.5 14.3 50.0 11.8 9.3 9.0 SE 6.8 6.9 37.2 47.0 7.2 1.4 0.4 0.3 SW 43.3 20.7 150.3 112.9 145.5 31.3 22.2 12.8 NW 44.4 16.0 143.2 19.9 51.4 33.5 9.1 6.4 Sep. 11 (at 12 d from start of anthesis) NE 71.1 3.1 21.9 16.7 17.4 3.7 3.2 2.0 SE 4.5 1.7 16.3 13.4 13.9 0.9 0.0 0.0 SW 25.3 14.3 97.4 15.9 10.8 8.7 13.1 17.0 NW 39.2 28.7 65.4 1.2 31.6 7.6 18.7 16.2
a Mean SD (n 2). b See Fig. 1 for direction.
As with the sunflower leaves (Table 2), the pollen reared on leaf discs treated with a pollen dose in density was higher at 1 m from the edge than at 80% acetone solution, corresponding to a pollen 0m, and no more than 20 grains were found at 5 m density of 1,000 grains per cm2 (d), both the sur- from the edge except for in SW-direction on Sep. 8 vival rate and proportion of second instar larvae (Table 3). were significantly higher than with treatment (c). This indicated that the toxicity of Cry1Ab in pollen Bt toxin level in pollen was considerably inactivated in the 80% acetone The Cry1Ab endotoxin level in pollen was solution. Therefore, we adopted the treatment (c) 29.3 5.7 mg/g (mean SD, n 3) in Event-176, as the bioassay method for evaluating the effect of and was not detected in non-transgenic corn Bt corn pollen on P. maha larvae. (N4640). Effect of Bt corn pollen on larval survival Evaluation of bioassay method In non-transgenic corn (N4640), 100 grains of The survival rate of P. maha larvae and the pro- pollen dose did not have a negative effect on the portion of larvae molting to the second instar did feeding and development of P. maha larvae, and not show a significant difference between (a) the the survival rate and the proportion of second in- intact O. corniculata leaf disc and (b) the leaf disc star larvae did not differ significantly among the treated with 80% acetone solution (Table 4). Sur- three pollen doses (20, 60 and 100 grains per cm2) vival rate and proportion of second instar larvae (Table 5). In transgenic corn (Event-176), larvae decreased significantly when larvae were reared on survived well at 10 grains per cm2 (95.8%), but the leaf discs at 1,000 grains per cm2 of Bt pollen after survival rate decreased significantly at more than pretreatment with 80% acetone solution (c), show- 20 grains (Table 5): the survival rate showed a sig- ing that Bt endotoxin in a pollen suspension affects nificant negative correlation with pollen dose larval survival. In contrast, when larvae were (r 0.965, p 0.01). The proportion of second in- Effect of Bt Corn Pollen on Non-Target Butterfly 157 a SD) 16.7 c 18.8 a 27.1 b 12.9 a a SD) 18.8 a 12.3 a 19.5 a second instar (%) Proportion to of larvae (mean 9.7 c 22.9 12.3 a 81.3 23.4 b 60.4 11.3 a 85.4 (d) (mean a SD) 12.3 a 81.3 9.7 a 91.7 12.3 a 83.3 ifferent at the 5% level by Tukey’s test. Tukey’s by at the 5% level ifferent ifferent at the 5% level by Tukey’s test. Tukey’s by at the 5% level ifferent at sixth day second instar (%) (mean 9.7 c 4.2 11.3 a 91.7 19.8 b 70.8 9.7 a 93.8 56 ) leaf with different treatments ) leaf with different a replicate SD) ) 17.9 b 4.2 9.7 a 93.8 16.7 a 77.1 2 Oxalis corniculata ollen dose No. P (grains/cm 23.8 b 20.8 7.2 a 89.6 34 a on wood sorrel ( on wood Survival rate (%) (mean Survival SD) 28.9 cd 21.7 d 60 12 91.7 33.9 ab 22.5 abc 20 12 95.8 11.3 a 40.1 bcd 100 12 91.7 hen exposed to transgenic and non-transgenic corn pollens at different doses to transgenic and non-transgenic cornhen exposed pollens at different (mean w 9.7 a 75.0 maha . P. 2 Pseudozizeeria maha a SD) 24.9 c 33.3 22.5 c 18.8 34.3 b 68.8 24.1 b 64.6 9.7 a 93.8 11.3 c 43.8 12 at sixth day second instar (%) (mean Survival rate (%)Survival Proportion to of larvae rate (%) Survival Proportion to of larvae 12 100 a 95.8 No. Larval survival rate of survival Larval replicate Larval survival rate of survival Larval . . e5 ransgenic corn (Event-176) Non-transgenic corn (N4640) e4 T bl bl replicate b Ta Ta ) 2 80 12 14.6 60 12 22.9 40 12 58.3 20 12 66.7 10 12 95.8 reatments 100 12 6.3 T ollen dose No. P (grains/cm in aqueous solution L + 80% acetone (80Ac) 12 100 a 100 a 100 a 100 a 95.8 b. c. L + 80Ac Bt1000 a. Leaf (L)d. L+ Bt1000 in 80Ac solution 12 100 a 12 100 a 100 a 100 a 97.9 100 a 95.8 Data were transformed to arcsin before statistical analysis. Means with different letters in the same column are significantly d letters in the same column are significantly Means with different transformed to arcsin before statistical analysis. Data were Bt1000 shows the corn pollen density of 1,000Bt1000 shows grains/cm Data were transformed to arcsin before statistical analysis. Means with different letters in the same column are significantly d letters in the same column are significantly Means with different transformed to arcsin before statistical analysis. Data were a a b 158 Y. SHIRAI and M. TAKAHASHI star larvae was highest at 10 grains, which was a Bioassay method significant difference between this and a more- A simple bioassay method was developed by than-60-grain dose pollen (Table 5), however, there using Event-176 that expressed high levels of was no significant correlation with the pollen dose Cry1Ab in pollen. Thus, pretreatment with a small (r 0.747, p 0.05). Thus, it suggested that a amount of 80% acetone solution allowed the prese- pollen dose of more than 20 grains per cm2 may lected pollen dose to be uniformly applied onto the have a harmful effect on the survival and develop- surface of a O. corniculata leaf disc, saving time in ment of P. maha larvae in the field. the bioassay. In addition, the procedure in which a weighable pollen dose is added to 1 ml of distilled water and 10 ml of this pollen suspension is pipet- DISCUSSION ted on the leaf disc (1-cm diameter) allows a more Actual pollen deposition density on leaves accurate evaluation of the effect of corn pollen de- Matsuo et al. (2002) have estimated, using a pending on the pollen dose. It is necessary to pre- mathematical model equation for maximal pollen pare the leaf disc on which low-pollen dose is de- dose, that approximately 4,000 grains or 2,000 posited in considering the actual pollen dose under grains per cm2 will be loaded at 10 m or 20 m from field conditions. the edge of cornfield, respectively. Although Ma- tsuo et al. (2002) assume a cumulative deposition Future procedures of risk assessment to non- dose for 18 d and the most suitable climatic condi- target lepidopteran species tions for the release of corn pollen, these estimated For Event-176, the larvae of P. maha were af- values are very different from the actual pollen fected at more than 20 grains per cm2 of pollen dose on the leaves of sunflower and black night- density (Table 5). Although we are unable to make shade (Tables 2 and 3). Corn tassels (male flowers) a direct reference because there is no data on have a 2-week flowering period, and most of the pollen deposition on O. corniculata leaves, the lar- pollen is shed within 3 d from the start of anthesis vae of P. maha may encounter risk within 5 m from (Williams, 2002). We considered that the pollen the edge of a cornfield. However, it can be con- dose on leaves in Tables 2 and 3 was not underesti- cluded that larvae of P. maha do not suffer risk due mated because this study covers the flowering sea- to the shedding of corn pollen at a distance of more son of corn, and it rained little throughout the dura- than 10 m from the field edge. For Event MON810 tion of the study (Table 1). The present results are and Bt-11, whose pollen has a low level of Cry1Ab in good accordance with the results of Pleasants et endotoxin (Andow, 2002), larvae of non-target lep- al. (2001) who have studied corn pollen deposition idopterans will not be affected within or at the edge dose on the leaves of the milkweed, the host plant of cornfields. In North America, it was concluded of the monarch butterfly, over 6 or 14 d from the that monarch butterfly larvae are not harmed in start of anthesis in and near a cornfields. They have cornfields of MON810 or Bt-11, but may be af- reported that the highest cumulative pollen density fected in limited areas near cornfields of Event-176 was ca. 100 to 220 grains per cm2 within cornfield, (Bartsch and Schuphan, 2002; Kaplan, 2002). falling to 50 to 70 grains at 1 m from the field edge, The registration of Event-176 was cancelled in 10 to 20 grains at 5 m and less than 20 grains at 2001 in the USA (Mendelsohn et al., 2003), and 10 m. Although we used different plant leaves, it is this transgenic corn event will be not marketed in unlikely that the pollen deposition dose on leaves Japan. Apart from Cry1Ab, some new transgenic exceeds 300 grains per cm2 even at a distance of 1 corn events for the protection of lepidopteran pests, to 2 m from the edge of the cornfield. In future expressing novel types of insecticidal protein (e.g. evaluation of the effect of corn pollen on non-tar- Cry1F and Vip3A), are scheduled for commercial get lepidopteran species, the present data could be cultivation (Mendelsohn et al., 2003; Moar, 2003). adopted as the pollen deposition dose in the field For Cry1F or Vip3A, the magnitude of risk to non- instead of the values estimated by Matsuo et al. target lepidopterans cannot be determined directly (2002). from the known information on Bt corn expressing Cry1Ab (MON810, Bt-11 and Event-176) (Wolt et al., 2003) because toxicity levels to a given insect Effect of Bt Corn Pollen on Non-Target Butterfly 159 species vary among types of Bt endotoxin (Herrero the Geranium bronze, Cacyreus marshalli Butler (Lepi- et al., 2002). In the risk assessment of these new doptera: Lycaenidae). Appl. Environ. Microbiol. 68: transgenic corns, endotoxin levels in corn pollen 4090–4094. Jesse, L. C. H. and J. J. Obrycki (2003) Occurrence of need to be evaluated by ELISA as a first step, and Danaus plexippus L. (Lepidoptera: Danaidae) in milk- if insecticidal protein is detected in pollen, larval weeds (Asclepias syriaca) in transgenic Bt corn agroe- bioassay could be conducted according to the pro- cosystems. Agric. Ecosyst. Environ. 97: 225–233. cedure shown in this study. From the actual pollen Kaplan, J. K. (2002) Bt corn not a threat to monarchs. deposition dose on leaves in the field, transgenic Agric. Res. (February 2002): 16–18. Knols, B. G. J. and M. Dicke (2003) Bt crop risk assessment corn pollen will pose no risk to the larvae of non- in the Netherlands. Nature Biotechnol. 21: 973–974. target lycaenid, P. maha, in the field when larval Losey, J. E., R. A. Hufbauer and R. G. Hartzler (2003) Enu- survival or development are not affected at 100 to merating lepidopteran species associated with maize as a 200 grains per cm2 of pollen density in the labora- first step in risk assessment in the USA. Environ. tory bioassay. However, if larval survival is signifi- Biosafety Res. 2: 247–261. cantly affected at a low pollen density of less than Losey, J. E., L. S. Rayor and M. E. Carter (1999) Transgenic 2 pollen harms monarch larvae. Nature 399: 214. 10 grains per cm , petition for the field cultivation Matsuo, K., S. Kawashima, M. Du, O. Saito, M. Matsui, K. of such transgenic corn may be rejected or post- Ohtsu, T. Ohkuro, T. Matumura and T. Mitamura (2002) poned due to a potential harmful risk to non-target Risk assessment for the dispersal of Bt-corn pollen to herbivores and environment. non-target lepidopterous insects. Bull. Natl. Inst. Agro- Environ. Sci. 21: 41–73 (in Japanese with English sum- ACKNOWLEDGEMENTS mary). Mendelsohn, M., J. Kough, Z. Vaituzis and K. Matthews We thank K. Matsuo (NIAES) for counting corn pollen dose (2003) Are Bt crops safe? Nature Biotechnol. 21: on leaves, K. Ohtsu (NIAES) for providing an authentic sam- 1003–1009. ple of Cry1Ab endotoxin, K. Abe (NIAES) for cultivation of Moar, W. J. (2003) Breathing new life into insect-resistant transgenic corn and collection of corn pollen, and M. Matsui plants. Nature Biotechnol. 21: 1152–1154. (NIAES) for valuable information on the rearing method of P. Nap, J. P., P. L. J. Metz, M. Escaler and A. J. Conner (2003) maha. The seeds of transgenic Bt-corn (Event-176, var. The release of genetically modified crops into the envi- Max.21) and non-transgenic corn (N4640) were provided by ronment Part 1. Overview of current status and regula- Syngenta Japan Co. Tokyo. This study was conducted at the tions. Plant J. 33: 1–18. request of the Research Council Secretariat, Ministry of Agri- Pleasants, J. M., R. L. Hellmich, G. P. Dively, M. K. 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