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Introduction:
The effects of Bacillus thuringiensis (Bt) corn, Zea mays, on nontarget organisms have received much attention since Losey et al. (1999) suggested in a correspondence to Nature that pollen from Bt corn could be hazardous to the larvae of the monarch butterfly, Danaus plexippus. In Losey’s laboratory investigation, young monarch larvae given no choice but to feed on milkweed, Asclepias syriaca, leaves dusted with pollen from a Bt corn hybrid ate less, grew more slowly, and had a significantly higher mortality rate than larvae feeding on leaves dusted with nontransgenic pollen. The authors questioned the environmental safety of Bt corn and called for scientific investigations. These preliminary findings were largely misrepresented by mainstream media before the potential impact of Bt corn pollen on monarch populations could be adequately assessed. Such reports have heightened public awareness, increased scrutiny of transgenic plants in terms of potential environmental impact, and intensified one of the most controversial and polarizing issues to face agricultural scientists in recent memory.
The corn hybrids in question were genetically modified to express an insecticidal protein derived from the bacterium B. thuringiensis. Bt provides yield protection from pest species such as the European corn borer, Ostrinia nubilalis, and some protection from other Lepidoptera (Pilcher et al. 1997) without the use of traditional insecticides or other management practices. The first transgenic plants were planted on a large scale in the United States in 1996 and have quickly been adopted by growers. Nearly 25 million acres of Bt field corn were planted in 1999, representing approximately 30% of total corn. Preliminary estimates for 2000 suggest this amount has deceased to 20 million acres, about 25% of total corn. More than a year has passed since initial concerns were expressed in the Nature correspondence. In response to these concerns, several researchers have begun detailed studies to evaluate the effects of Bt pollen on monarch larvae. This chapter provides an overview of these investigations and significant related events.
Bt Corn Registration and Nontarget Insects:
The Environmental Protection Agency (EPA) has required all Bt corn registrants to provide information on possible effects of Bt corn on honey bees and other beneficial nontarget insects, such as lady bird beetles and green lacewings. Examples of tests required by registrants for approval of Bt corn are listed at the EPA web site. Additionally, EPA has provided fact sheets for all Bt corn events that have been registered and for those that are seeking registration. In the EPA required non-target insect tests, insects were fed high concentrations of Bt protein and no negative effects were observed. The toxicity of Bt proteins expressed by transgenic corn to larval stages of butterflies and moths is well known (MacIntosh et al. 1990). Many studies, particularly those conducted on the extensive use of Bt sprays in forests for gypsy moth control, have shown that Bt Cry proteins can adversely affect nontarget Lepidoptera (Miller 1990; Johnson et al. 1995). Field data from these studies indicated a temporary reduction in lepidopteran populations during prolonged Bt use, although widespread irreversible harm was not apparent (Hall et al. 1999). Based on such information, the EPA made the assumption that B. thuringiensis is a hazard to all Lepidoptera, but that exposure from agricultural uses of Bt was not expected to be as high as in forest spraying. Bt corn also was not expected to impact nontarget butterflies and moths.
Toxicity Studies:
The amount of pollen dusted onto the milkweed leaves in the Losey et al. (1999) study was not quantified, and as a result, the dose of Bt protein consumed by the larvae could not be quantified. To formulate a quantitative risk assessment, the level of toxicity must first be determined. Generally dose-response studies are conducted to determine estimates of the LC50, or lethal concentration that kills 50% of tested insects. Dose-response relationships of four Bt proteins were conducted by Blair Siegfried (University of Nebraska) with monarch neonates (newly hatched larvae). Neonates were exposed for 7 days to purified Bt toxins incorporated into an artificial diet. All toxins currently available in Bt corn (Cry1Ab, Cry1Ac, and Cry9C) and one under development (Cry1F) were tested. Results of these studies indicate that monarch larvae are highly sensitive to certain Bt toxins, whereas others are relatively nontoxic. Monarch neonates were most sensitive to Cry1Ab and Cry1Ac, even more so than European corn borer neonates, although direct comparisons have yet to be conducted. In contrast, Cry9C and Cry1F were considerably less toxic; therefore, risks associated with plants expressing one or the other of these proteins are likely to be reduced relative to the Cry1Ab and Cry1Ac events. The commercially available Cry1Ac event, DBT-418, is in the process of being phased out, and has received little further attention. Consequently, most of the exposure questions have focused on the Cry1Ab events: 176, BT11, and MON810.
Bioassays conducted by Richard Hellmich (USDA–ARS) and Mark Sears (University of Guelph) with monarch neonates fed milkweed leaves treated with pollen collected from Bt corn plants generally support Siegfried’s findings. It should be noted, however, that susceptibility to Bt pollen varied among events known to express the same toxin. Events 176 and MON810 both express the Cry1Ab toxin, but 176 expresses at higher levels due to a pollen-specific gene promoter (Koziel et al. 1993). Event 176 pollen affected neonates by increasing mortality and reducing body weight at pollen concentrations substantially lower than that of event MON810. Wraight et al. (2000) found that black swallowtail larvae, Papilio polyxenes, also were more sensitive to 176 pollen compared with MON810 pollen, based on field exposures.
Preliminary data suggest that pollen from event 176, compared with all the other Bt events, may pose the highest risk to monarch larvae. However, the potential risks to monarch populations are likely to be minimal, because sales of 176 corn hybrids in 1999 represented only approximately 2% of total corn. Fields of 176 corn represented very small portions of the total land area of each state (Figure 1).
Exposure Studies:
Although toxicity of Bt pollen and the toxins expressed by pollen can be clearly documented and could represent a hazard, the risk to monarch populations from exposure to Bt corn pollen is also a function of exposure. If exposure to the toxin under field conditions is minimal, then even highly toxic materials pose little risk. For monarchs to be exposed and potentially affected by corn pollen, larval development must coincide with corn anthesis (pollen shed). Throughout much of the Midwest, corn anthesis occurs during a short period (3-7 days) in July. Exposure to the pollen, however, may be longer depending on the duration of pollen availability on milkweed and the rate of Bt protein degradation. In 1999, anthesis overlapped with larval development in Iowa, but according to observations made by John Foster (University of Nebraska) in Nebraska and Galen Dively (University of Maryland) in Maryland, the overlap was of short duration. Dennis Calvin (Pennsylvania State University), Joe Russo (ZedX, Inc.), and Orley Taylor (University of Kansas) are developing models to identify areas where corn anthesis and monarch larval development are likely to occur simultaneously. During the 2000 summer, monarch observations from corn-growing areas will provide information to validate these predictions. .
In areas where monarch larval stages overlap with corn anthesis there are a number of questions associated with pollen deposition and movement and their joint effect on exposure. Determining how much pollen is shed and how far the pollen drifts is necessary for an accurate assessment of exposure. Experiments were conducted in the summer of 1999 to measure deposition of corn pollen on milkweed plants in and near cornfields. Results from four independent laboratories (John Foster; Galen Dively; Mark Sears, University of Guelph; and John Pleasants, Iowa State University) indicate that pollen deposition sharply decreases at short distances from cornfields. Most pollen deposition occurs within the cornfield, and pollen quantities found on milkweed leaves and the proportion of leaves with pollen decreased rapidly 2 to 3 m from the fields. Similar pollen deposition results were reported by Raynor et al. (1972) and Wraight et al. (2000). This steep deposition gradient is influenced by the large size of corn pollen, typically 90 to 100 micrometers in diameter. Pleasants and Hellmich examined pollen deposition within and at various distances from cornfields in Iowa by using glass slides coated with glycerin to trap pollen deposits, and pairs of milkweed leaves termed “boutonnieres.” The concentration of pollen deposited on milkweed leaves followed the same pattern with distance and wind direction as the concentration on glycerin-coated slides, although the total amount deposited on leaves was much less than on slides. On average, milkweed leaves retained only 30% of the pollen available at a given sampling location. Also, a rain event during one of the experiments removed 90% of the pollen from milkweed leaves.
Another important factor in assessing exposure of monarch larvae to Bt pollen is milkweed distribution. Milkweeds in or adjacent to cornfields are likely to have more corn pollen on them than milkweeds in other habitats. Thus, it is important to assess the relative numbers of milkweeds in different habitats to fully assess the potential impact of Bt corn pollen on monarch populations. In the summer of 1999 Doug Buhler (USDA–ARS) and Robert Hartzler (Iowa State University) conducted a comprehensive survey of common milkweed plants across Iowa. Common milkweed was found in 71% of the roadsides and approximately 50% of the corn and soybean, Glycine max, fields (Hartzler and Buhler 2000). Corn and soybean fields had 85% fewer patches than roadsides. Conservation reserve program (CRP) fields had the greatest average area infested. While common milkweed was frequently found in corn and soybean fields, average frequency and patch size were much greater in noncrop areas. This survey will be modified in the summer of 2000 to evaluate the stability of common milkweed populations and to better document common milkweed populations in CRP fields. There are several important questions that remain regarding the distribution of milkweed in different habitats across the Midwest and the relative importance of milkweed in these habitats as hosts for developing monarch larvae.
Behavior of monarch larvae and adults also can influence exposure to Bt pollen. Larval preference for milkweed leaves with pollen would increase exposure to Bt protein, but an avoidance of such leaves would have the opposite effect. Likewise, Bt exposure would be influenced if females had an oviposition preference for or against milkweed plants in or near cornfields.
Preliminary choice tests conducted by Hellmich and Leslie Lewis (USDA–ARS) suggest that monarch larvae are influenced by the presence of pollen, although their behavior changes with pollen concentrations. When presented with a choice of leaf discs without pollen and leaf discs with high amounts of pollen, more larvae were found on the discs without pollen. However, when presented with leaf discs without pollen and leaf discs with small amounts of pollen, more larvae were found on the leaves with pollen. Studies are being conducted to determine the importance of larval choice under field conditions and to determine the role, if any, that feeding cessation has when larvae encounter Bt protein.
Corn hybrids are taller than milkweed plants when corn anthesis occurs, but it is not known whether female oviposition is influenced by corn plant height. Coordinated field studies are being conducted in Minnesota (Karen Oberhauser and Michelle Prysby), Iowa (Pleasants and Hellmich), New York (John Losey), Maryland (Dively), and Ontario (Sears) to determine the distribution of monarch life stages found in cornfields, other crop fields, roadsides, pasture, and set-aside land. These studies should determine whether female oviposition preference is influenced by habitat and the importance of milkweed plants growing in cornfields to monarch development.
Nontarget Insect Meetings:
Preliminary results from the 1999 laboratory and field studies were first reported in Chicago, IL at the November 1999 Monarch Conference. This conference served as an important forum for researchers, regulators, environmental advocacy groups, and industry. It was clear, however, that the results presented at this meeting were preliminary and incomplete. As a result of this meeting, a consortium of researchers was formed to identify gaps and overlaps in the data, promote an open exchange of information, and provide a coherent research agenda. The Chicago conference was followed in February 2000 by an USDA-sponsored Monarch Research Workshop in Kansas City, MO. A steering committee, including Adrianna Hewings (USDA–ARS), Eldon Ortman (Purdue University), Mark Scriber (Michigan State University), Eric Sachs (Monsanto), and Margaret Mellon (Union of Concerned Scientists), was formed to provide guidance for the workshop and subsequent consortium activities. The goal of the workshop was to identify research priorities regarding Bt corn and monarch butterflies and establish cooperation among researchers. Approximately 40 government, academic, and industry scientists participated in the workshop. Attendees identified short- and long-term research priorities, which were summarized by the steering committee. Short-term research objectives included the following: 1) determine importance of cornfields in monarch population production; 2) continue laboratory bioassays to further define dose-response relationships and sublethal effects; 3) expand land-use surveys to determine milkweed distribution, and abundance; 4) determine monarch distribution, abundance and survival in Bt and non-Bt corn production systems; and 5) collect data to field verify models that predict co-occurrence and dose-mortality response of monarch larvae and corn anthesis. A request for proposals based on these priorities was announced April 7, 2000. USDA–ARS and industry, through an unrestricted gift from the Agricultural Biotechnology Stewardship Technical Committee, each made $100,000 available for research projects outlined by the consortium.
December 1999, a Scientific Advisory Panel was convened to provide advice, information, and recommendations to EPA regarding characterization and nontarget organism data requirements for protein plant-pesticides. The panel recommended that selection of nontarget insects should be made on a case-by-case basis and that species that have an ecological association with the crop plant or target insect should be considered. These species could include nontarget relatives of the target pest (e.g., nontarget Lepidoptera). The EPA is taking steps to address issues raised by the panel to more adequately address issues surrounding effects of nontarget organisms for current and future registrations of transgenic plants.
Summary:
The ability to transform crop plants to express the insecticidal toxins from B. thuringiensis is likely to have profound effects on the future of pest management. The benefits of the technology in terms of yield protection and reduced environmental disruption relative to synthetic insecticides must be balanced in terms of the uncertainty associated with risk to nontarget organisms, such as the monarch butterfly. The issue has drawn attention to an important aspect of genetic engineering. More attention needs to be directed at clarifying risk assessment and communicating that information, particularly to non-scientific audiences.
The consortium of researchers, industry, and environmental advocacy groups that has been assembled to address concerns related to the monarch butterfly and Bt corn represents an unparalleled level of cooperation and is indicative of the product stewardship that is essential for the full benefits of the technology to be realized. One of the goals of the consortium is to develop high quality research data so that decision making is based on sound science.
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Article Copyright Academic Press, London 2000
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