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 (18ϫ18 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 (7ϫ7 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 Ϫ30°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 Ϫ30°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.