Published online on September 14, 2001, 10.1073/pnas.171315698
Agricultural Sciences
Effects of exposure to event 176 Bacillus
thuringiensis corn pollen on monarch and black swallowtail
caterpillars under field conditions
A. R.
Zangerl,
D.
McKenna,
C. L.
Wraight,
M.
Carroll,
P.
Ficarello,
R.
Warner, and
M. R.
Berenbaum*
Department of Entomology, University of Illinois at
Urbana-Champaign, Urbana, IL 61801
Contributed by M. R. Berenbaum, June 21, 2001
 |
Abstract |
The widespread planting of corn genetically modified to produce
Bacillus thuringiensis endotoxin has led to speculation
that pollen from these fields might adversely affect nearby nontarget lepidopterans. A previous study of Bt corn engineered
with Monsanto event 810 failed to detect an effect of pollen exposure
on the black swallowtail, Papilio polyxenes, in either
the field or the laboratory. Here, we report results of a field study
investigating the impact of exposure to pollen from a Bt
corn hybrid containing Novartis event 176 on two species of
Lepidoptera, black swallowtails and monarch butterflies, Danaus
plexippus. Nearly half of the 600 monarch larvae died within
the first 24 h; this and subsequent mortality was not associated
with proximity to Bt corn and may have been due in part
to predation. Survivorship of black swallowtails was much higher than
that of the monarchs and was also independent of proximity to the
transgenic corn. However, despite five rainfall events that removed
much of the pollen from the leaves of their host plants during the
experiment, we observed a significant reduction in growth rates of
black swallowtail larvae that was likely caused by pollen exposure.
These results suggest that Bt corn incorporating event
176 can have adverse sublethal effects on black swallowtails in the
field and underscore the importance of event selection in reducing
environmental impacts of transgenic plants.
| |
Introduction |
With the exception of
herbicide-tolerant soybeans, Bt corn (Zea mays
engineered to express genes from the soil bacterium Bacillus thuringiensis that encode the insecticidal protein toxins Cry1Ac, Cry1Ab, or Cry9C) is the most widely grown transgenic crop plant in the
United States. In 1999, Bt corn was planted on 9.6 million hectares (1). The principal target species for Bt corn is
the European corn borer (ECB), Ostrinia nubilalis, one of
the most damaging pests of corn in North America
(http://www.extensionumn.edu/Documents/D/C/DC7055.html). Losses to ECB damage and costs of control range upward of $1 billion annually in the United States. In addition to direct damage, ECB damage
leaves corn vulnerable to infection by Fusarium fungi; these
pathogens can produce highly toxic fumonisins, which pose a risk to
human health if ingested.
Although Bt corn has been touted as an environmentally
friendly alternative to the synthetic organic insecticides
traditionally used for ECB control in sweet corn (including permethrin,
bifenthrin, lambda-cyhalothrin, and methyl parathion) (2), concerns
have been raised that there may be adverse effects of Bt
corn use on nontarget lepidopterans and their consumers (3). In a
laboratory feeding study, Losey et al. (4) demonstrated that
exposure to Bt corn pollen can cause mortality in neonate
monarch caterpillars (Danaus plexippus). Despite the fact
that the authors cautioned that "it would be inappropriate to draw
any conclusion about the risk to monarch populations in the field based
solely on these initial results," the study created a widespread
perception of risk, particularly among nonscientists (5). In a second
study, Hansen-Jesse and Obrycki (6) fed milkweed foliage, which was "naturally dusted" under field conditions with pollen from
Bt corn, to monarch caterpillars in laboratory feeding
trials; they reported significantly greater mortality of larvae that
consumed foliage contaminated with Bt pollen, although no
dose-dependent effect of pollen concentration was observed.
To date, the only published study done to examine the consequences of
exposure to Bt corn pollen on nontarget lepidopterans in the
field is Wraight et al. (7). In this study, which dealt not
with D. plexippus but rather with Papilio
polyxenes, the black swallowtail, no mortality could be directly
attributable to exposure to MON810 corn pollen under field conditions.
How representative P. polyxenes is of nontarget
lepidopterans that live alongside cornfields is an open question. In a
companion laboratory experiment, however, these authors demonstrated
that P. polyxenes is sensitive to pollen from Novartis event
176, which contained 40-fold higher concentrations of endotoxin than
does MON810.
Environmentalists as well as government regulators are calling for more
detailed studies on possible nontarget impacts of Bt corn.
In the Federal Insecticide, Fungicide and Rodenticide Act Scientific
Advisory Panel Report No. 99-06, released February 4, 2000 (http://www.epa.gov/scipoly/sap/1999/december/report.pdf), "Characterization and non-target organism data requirements for protein plant-pesticides," the Panel concluded that current
nontarget testing requirements were inadequate, in that they were
limited in terms of species numbers, and called for "additional
research . . . on the various possible effects of plant pesticidal
proteins on non-target insects." Here, we report the results of a
study comparing responses of two different nontarget species with
larval ecologies that place them at risk of exposure; both the black swallowtail P. polyxenes and the monarch caterpillar
D. plexippus feed on weedy forbs that are frequently found
in or around cornfields throughout the Midwest. Moreover, for the first
time, we document sublethal effects of Bt corn pollen on
growth and development of P. polyxenes in the field.
| |
Materials and Methods |
Host Plants.
The host plant selected for testing P. polyxenes
susceptibility to Bt corn pollen was wild parsnip,
Pastinaca sativa. P. sativa grows extensively along field
edges throughout central Illinois and is available as a food plant
during the period when corn sheds pollen. Plants were grown in the
greenhouse from seed collected in the field in Champaign County, IL and
sown in plastic pots measuring 17 cm in diameter and 21 cm in height
and containing a mixture of one part soil, two parts peat, and two
parts perlite. At the time of the experiment, these plants were
rosettes with several large compound leaves.
The host plant selected for testing D. plexippus
susceptibility to Bt corn was Asclepias syriaca,
like parsnip, a conspicuous element of the flora found at field edges
in central Illinois (8). All but five of the milkweed plants used in
the experiment were grown in the greenhouse from locally collected
seeds. Seeds were sown in pots and soil identical to those used for the
parsnips. At the time of the experiment, these plants were between 60 and 90 cm tall. Several of the milkweeds became diseased, as evidenced by a darkening of the leaves, and were discarded. As a result, there
were five fewer seed-grown plants than the 25 plants required for the
experiment. Consequently, additional milkweeds were obtained by
transplanting A. syriaca in the vicinity of the experimental plot to pots containing field soil.
Insects.
Black swallowtail eggs were obtained from 23 females caught in central
Illinois shortly before the start of the experiment. The females were
caged together with wild parsnips to elicit oviposition. Monarch eggs
deposited on milkweed foliage were provided by Dr. Patrick Hughes from
a colony maintained at the Boyce Thompson Institute (Ithaca, NY).
Although the use of insects from a laboratory colony may introduce
artifacts due to unusual behaviors or physiology resulting from
continuous rearing, the requirement for large numbers of eggs on a
particular date precluded use of local wild-caught females, which were
not sufficiently abundant at the time of the experiment to provide a
large single cohort of neonates.
Field Experiment.
Field plot design.
A 30 × 30-m plot of Max 454 Bt corn, which
contains Novartis event 176, was planted in late May with rows oriented
north-south at the University of Illinois Phillips Tract research
area, located 1.5 km northeast of Urbana. To ensure that we had an edge
consisting of plants of uniform and typical size, an 8-m swath of the
corn was mown along the north side of the plot two weeks before
initiation of the experiment.
The corn first began to shed pollen on July 24, 2000. The following
day, we placed 20 potted parsnips and 25 potted milkweeds, numbers
sufficient to accommodate the larvae available, in an array on the
north side of the plot. The first row of plants, consisting of four
parsnips and five milkweeds, was situated 0.5 m from the edge of
the corn. Additional rows were positioned 1, 2, 4, and 7 m from
the corn. Except for the extra milkweed plant, within a row, parsnips
and milkweeds were paired. Within a row, the spacing between pairs and
between a pair and the lone milkweed was 1 m.
Rate of pollen deposition.
Commencing on July 26 and on each subsequent day between 9:00 am and
noon for the duration of the experiment, we sampled pollen from two
randomly selected parsnips and milkweeds at each distance from the
cornfield. The pollen was sampled by applying 2-3 drops of a mixture
of 1 part Duco cement (Devcon Consumer Products, Rivera Beach, FL) and
3 parts acetone onto a leaf. After the glue dried, it was carefully
peeled off with forceps, removing all of the pollen grains while
maintaining their spatial pattern. This method of pollen sampling is
superior to other methods of pollen monitoring in that it allows
accurate determination of pollen densities on the foliage without
removing foliage, which may alter plant chemistry. Moreover, the method
also prevents loss of pollen through disturbance and permits grains to
be stained in their original dispersion pattern.
After the peel was removed, the location on the leaf that was sampled
was marked with a small "x" in black indelible ink to ensure that
it would not be subsequently resampled. These "pollen peels" were
brought to the laboratory and stained with a dye consisting of 5 ml of
glycerol, 10 ml of ethanol, 15 ml of water, and enough crystals of
basic fuchsin to render the staining solution a deep red. The peels
were rinsed in distilled water, and the pollen grains were counted at
40× magnification. Counts were converted to number of grains per
cm2 of leaf area and averaged for the two samples
per host plant at each distance from the cornfield.
Caterpillar performance.
Larvae of both species were placed onto plants in the field when
sufficient numbers of eggs hatched to produce a large cohort of
neonates. Neonate monarchs, 24 to a plant, were placed on a fully
expanded leaf near the top of 24 of 25 plants, one of the transplanted
milkweeds having died before egg hatch. All other milkweed plants
remained healthy and vigorous throughout the duration of the
experiment. In total, 600 larvae were put in place on July 28, 4 days
after the onset of pollen release. On the following day, 15 swallowtail
neonates were placed on a single leaf of each of 20 parsnip
plants for a total of 300 caterpillars. The number of live
larvae remaining each day was recorded for a total of 6 days for
monarchs and 5 days for swallowtails. We modified our census procedure
on the second day, after observing a nearly 50% decline in monarchs
24 h after placement of the larvae. Because the decline appeared
to be unaffected by proximity to the corn, we suspected that predation
was the cause. Accordingly, as part of the daily protocol, we continued
to census caterpillars and also recorded the number and type of
predaceous arthropods on each plant. After predators were identified
and counted, they were destroyed.
The final census date, August 3, coincided with the cessation of pollen
shed and followed by one day the last of five rainfall events, which
took place on July 28, 29, 30, 31, and August 2. On the final census
day, all larvae were collected and weighed to the nearest milligram in
the laboratory. Because the distribution and variation in the larval
mass data for black swallowtails did not conform to the requirements
for analysis of variance, a Kruskal-Wallace analysis was performed to
determine whether proximity to the cornfield affected larval mass
(SPSS, Chicago). Survivorship curves in the field were compared as a
function of distance by Kaplan-Meier analysis (survival procedure in SPSS).
Laboratory bioassay.
As reported in an earlier study (7), four pollen concentrations,
10,000, 1,000, 100, and 10 grains per cm2 of
Bt pollen, together with an acetone control were bioassayed in the summer of 1999 against first instar black swallowtails in the
laboratory. In that study, the results for only a single concentration
of Max 454 pollen were reported. In this study, we report an
LD50 for Max 454 based on all five concentrations. The
LD50 of the pollen was determined by probit
regression (SPSS), and survivorship curves at different doses were
compared by Kaplan-Meier analysis (survival procedure in SPSS).
| |
Results |
Rates of Pollen Deposition.
Pollen counts varied considerably, depending on the location of the
sample within a leaf. Pollen was most prevalent along major veins and,
on milkweed leaves, particularly along the midvein. Despite the high
variation in pollen densities from sample to sample, patterns of pollen
distribution observed in this study (Fig.
1) were consistent with those previously
reported (6, 7, 9), in that the greatest amounts of pollen were
deposited in close proximity to the cornfield.
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Fig. 1.
Pollen densities on foliage of milkweeds (A. syriaca)
and wild parsnips (P. sativa) as a function of time and
distance from a stand of event 176 Bt corn. In each
case, larvae were placed on the plants on the earliest date indicated.
Rainfalls of 0.20, 0.43, 0.10, and 0.10 cm occurred on July 28, 29, 30, and 31, respectively. An additional rainfall of 1.56 cm occurred on
August 2 after the last pollen sampling and the day before the final
larval census.
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Pollen densities before placement of the larvae on the plants (July 27, data not shown in Fig. 1) were higher than those on the day the larvae
were put in place. For example, on milkweed foliage, the highest
average density of pollen was 260 grains/cm2 on
plants 0.5 m from the corn and 170 grains/cm2 on plants located 1 m away from
the corn.
On parsnip foliage, the highest average pollen density was 320 grains/cm2 0.5 m from the corn. Remarkably
high densities (180 grains/cm2) were observed
as far as 2 m from the corn. However, because of rain events, the
test larvae did not experience these higher pollen levels. In fact,
pollen densities declined dramatically after rain events (Fig. 1).
Caterpillar Performance.
Mortality was most pronounced among the monarch larvae. After 6 days in
the field, fewer than 7% of the larvae survived (Fig. 2). The decline was greatest during the
first 24 h, and the rate of decline slowed after we began
systematically removing predatory arthropods. Despite clear differences
in exposure to pollen as function of distance (Fig. 1), survivorship
curves did not differ significantly according to distance (log rank
statistic = 1.22, P = 0.269). Proximity also did
not affect mass of the surviving larvae (ANOVA F = 0.528, df = 4, 17, P = 0.716), although, with only
22 of the 600 monarch larvae surviving, statistical power was extremely
low for this analysis.
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Fig. 2.
Survivorship of monarch (D. plexippus) and black
swallowtail (P. polyxenes) larvae as a function of
distance from a field of event 176 Bt corn.
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Mortality of black swallowtail larvae was noticeably less than that of
monarchs (Fig. 2), and, as was the case for the monarchs, mortality was
not affected by proximity to corn; there were no significant
differences among survivorship curves (log rank statistic = 1.62, P = 0.203). However, larval masses were significantly negatively affected by proximity to the corn (Fig.
3). Larvae 7 m from the corn
attained a biomass that on average was three times that of larvae
located 0.5 m from the corn. The best-fitting regression between
mean larval mass and proximity to corn was linear (F = 30.3, P = 0.012) and accounted for 91% of the
variation.
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Fig. 3.
Larval masses of the black swallowtail (P. polyxenes) at
different distances from a field of event 176 Bt corn.
The white lines within the boxes are the means. The boundaries of the
boxes represent the 25th and 75th percentiles, and the boundaries of
the whiskers represent the 10th and 90th percentiles.
 , outliers (one outlier value of 51.8 in the
7 m position is not shown). Larval masses differed significantly
among distance treatments (Kruskal-Wallace  2 = 60.5, P < 0.001). The regression of mean larval
mass against distance was also significant (P = 0.012, y = 1.36x + 5.23). Sample
sizes for distances from 0.5 to 7 m were 39, 26, 25, 36, and 46, respectively.
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Despite daily removals, the frequency of predaceous arthropods was
remarkably constant and, based on cumulative totals, remained constant
because of a steady influx of individuals (Fig.
4). On two sampling dates, predator
numbers were significantly greater on milkweed plants than on parsnip
plants (Fig. 4); differences in predator number may have contributed to
the lower survivorship of monarchs in the field. By far the most
abundant predators were adult and larval coccinellids (Fig.
5). They were found on both host plants
but were most numerous on milkweeds in the vicinity of the apical
meristem, where aphids had become established and where many of the
monarch larvae moved after being placed on a fully expanded leaf near
the top of the plant. Very few acts of predation were observed during
the sampling periods; on one occasion, an immature Podisus
maculiventris was observed and photographed consuming a monarch
larva.
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Fig. 4.
Daily and cumulative totals of arthropods found and removed from
milkweeds and wild parsnips during the experiment. Dates with asterisks
indicate significantly higher numbers of predaceous arthropods on
milkweeds (2 values = 11.26 and 8 for July 28 and
29, respectively; both values have probabilities less than 0.05).
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Fig. 5.
Frequency of taxonomic groups found and removed from both milkweeds and
wild parsnips during the experiment. Asterisks denote groups known to
be generalist consumers of lepidopteran larvae.
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Laboratory Bioassay.
The laboratory bioassays of event 176 pollen revealed significant
mortality at doses of 100 grains/cm2 and higher
(log rank test P = 0.0405 for comparison of the 100 grains/cm2 dose with the control) (Fig.
6). The LD50 for
Bt pollen in this bioassay was 613 grains/cm2, with a 95% confidence interval
between 299 and 1151.
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Fig. 6.
Survivorship of first instar black swallowtails administered different
doses of event 176 pollen in the laboratory. The only survivorship
curve that does not differ from control is the 10 grains/cm2 (log rank test P = 0.263).
Pollen was suspended in acetone and applied to leaf material. The
control consisted of addition of acetone only. For details of the
bioassay method, see Wraight et al. (7).
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| |
Discussion |
In 1999, Wraight et al. (7) conducted a study designed
to detect effects of event MON810 pollen on black swallowtails. That
study failed to detect an effect of Bt pollen on either
survivorship or larval mass in the field or on survivorship in the
laboratory. However, a laboratory bioassay of event 176 pollen
demonstrated that this pollen was toxic at high doses to black
swallowtails. The bioassay reported in this present study (Fig. 6)
indicates that concentrations of event 176 pollen as low as 100 grains/cm2 cause significant mortality in black
swallowtails. Given the wide confidence interval, the
LD50 for black swallowtails, 613 grains/cm2, is not substantially different from
the LD50 of 389 grains/cm2 reported for monarchs (9).
The results of this study suggest that pollen from Bt corn
varieties engineered with the 176 event may have sublethal effects on
black swallowtails feeding on host plants situated outside of
cornfields. The cause of the reduction in larval mass as a function of
proximity to the Bt corn (Fig. 3) is most likely toxicity because of the ingestion of transgenic pollen grains. Alternative explanations not involving Bt pollen fail to account
satisfactorily for the observed pattern of larval masses. Possible
explanations involve modification of the thermal environment by the
corn. A stand of corn may affect the thermal environment outside its
boundaries via shading and mass flow of transpirationally cooled air.
Because the experiment was situated on the north side of the field,
where the flow of pollen outside of the field was expected to be
greatest (prevailing summer winds are from the south and southwest), a shadow cast by the corn could have fallen on larvae near the corn. By
virtue of their early instar black color and habit of feeding exposed
on the upper sides of leaves, black swallowtail body temperatures are
likely to be affected by exposure to direct sunlight. However, at the
time that the experiment was performed, the sun was close to its
zenith, and the broken shadow cast by the stems and leaves extended no
farther than 0.7 m from the field's edge. Thus, the impact of
shading could not have affected the larvae at 1 m and beyond. A
reanalysis of larval masses omitting the 0.5-m data remained highly
significant (Kruskal-Wallace 2 value = 30.766, P < 0.001), ruling out the influence of
shading on reduced larval mass.
Another possible explanation for reduced growth is that evaporatively
cooled air from the corn caused a temperature gradient extending from
the borders of the corn that in turn caused differences in growth rate.
This explanation also appears unlikely to account for our results.
Three days after completion of the experiment, we measured air
temperatures at 0.5, 1, 2, 4, and 7 m along ten transects within
the experimental plot. Temperature was measured 30 cm above the ground
with a TH-65 thermocouple thermometer (Wescor, Logan, UT). We did find
a significant temperature gradient increasing away from the field
(y = 0.262x 
0.016x2 +25.98, F = 7.76, df = 47, P = 0.004). However, the difference in mean temperature between 0.5 m and 7 m was only 0.94°C.
For a single degree difference to cause a 3-fold increase in growth, the Q10 for growth of the black swallowtail would
have to have been on the order of 60,000. Growth
Q10 levels based on data from Knapp and Casey
(10) were on the order of 2 and 5, respectively, for two other
lepidopteran species, Lymantria dispar and Malacosoma americanum. Effects on thermal environments and other potential effects of cornfields not involving pollen are further discounted by
the lack of any effect on larval mass of a different and less toxic
form of Bt corn (7).
The effect of the event 176 Bt pollen in this study is
presumably less than the potential effect that would have been observed had repeated rainfalls not washed pollen off of the foliage. Only once
during the swallowtail experiment did pollen levels exceed 60 grains
per cm2 beyond 0.5 m (Fig. 1), whereas,
before the experiment and before the rainfalls, average pollen
densities were three times higher as far as 2 m from the corn (192 grains/cm2). Consequently, the effects observed
in this study must be considered conservative.
Whether the observed reduction in larval mass was due to direct
toxicity of ingested pollen or to antifeedant effects of pollen exposure (e.g., ref. 6) is unclear. Also unresolved are the long-term
consequences of larval mass reduction over the life of the caterpillar.
Hansen-Jesse and Obrycki (6) suggest that age may influence
susceptibility of monarch larvae to pollen ingestion. Moreover, because
the greatest amount of all food ingested over the course of caterpillar
development is consumed in the ultimate instar, caterpillars
experiencing developmental delays early in life may be able to
compensate if they subsequently consume uncontaminated foliage.
Notwithstanding, this study documents sublethal effects of event 176 Bt corn pollen on a nontarget lepidopteran outside of a
cornfield. Wraight et al. (7) suggested that nontarget
impacts may be manageable by careful event selection. To some extent, this approach is already underway. During the 2000 growing season, event 176 corn comprised less than 1% of total U.S. acreage (Jeff Stein, Novartis, personal communication), and reregistration of the
technology with the U.S. Environmental Protection Agency is not
anticipated
(http://www.nk.com/infosilo/news/release.cfm?releaseId = 113&sKeyword = newsrelease).
That there may be nontarget impacts of Bt corn is not in
itself surprising; there are nontarget impacts of any pest management approach. Risks and benefits of Bt corn must be evaluated
relative to alternative methods of management (3). A recent National Academy of Sciences study (NAS 2000) emphasizes the need for
maintaining a "diverse toolbox" for pest management; careful use
of tools, which includes use of the most appropriate tool for each
situation, can help to preserve their utility as well as to maintain
environmental quality.
| |
Acknowledgements |
We thank Dr. Patrick Hughes for supplying us with monarch eggs,
Robert Dunker for planting and maintaining the Bt
cornfield, and Steve Buck, Katrina Lustofin, Xianchun Li, and Daniel
Skirvin for technical assistance. We also thank Kevin L. Steffey and
Fred Gould for their careful review of the manuscript. This study was supported by funds from the University of Illinois Foundation and the
University of Illinois at Urbana-Champaign Center for Advanced Study
(to M.B.).
| |
Abbreviation |
ECB, European corn borer.
| |
Footnotes |
*
To whom reprint requests should be addressed. E-mail:
maybe{at}uiuc.edu.
| |
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Abstract
Introduction
