French.cornpest.ca

Increased survival of western corn rootworm on
transgenic corn within three generations of on-plant
greenhouse selection
Lisa N. Meihlsa, Matthew L. Higdonb, Blair D. Siegfriedc, Nicholas J. Millerd, Thomas W. Sappingtond, Mark R. Ellersiecke,
Terence A. Spencerc, and Bruce E. Hibbarda,b,1
aDivision of Plant Science, 205 Curtis Hall, University of Missouri, Columbia, MO 65211; bUSDA-ARS, 205 Curtis Hall, University of Missouri, Columbia, MO65211; cUniversity of Nebraska-Lincoln, Department of Entomology, 202 Plant Industry Building, Lincoln, NE 68583; dUSDA-ARS, Genetics Laboratory, IowaState University, Ames, IA 50011; and eAgricultural Experiment Station Statistician, 307E Middlebush, University of Missouri, Columbia, MO 65211 Edited by May R. Berenbaum, University of Illinois at Urbana-Champaign, Urbana, IL, and approved October 16, 2008 (received for review June 10, 2008) To delay evolution of insect resistance to transgenic crops produc-
H. virescens or most other major lepidopteran pests targeted by ing Bacillus thuringiensis (Bt) toxins, nearby ‘‘refuges'' of host
Bt crops (3).
plants not producing Bt toxins are required in many regions. Such
As in the case of Cry1Ac targeting H. zea, the Bt corn, Zea refuges are expected to be most effective in slowing resistance
mays L., currently registered for control of corn rootworms when the toxin concentration in Bt crops is high enough to kill all
(Diabrotica spp.) is not high-dose, but rather is considered or nearly all insects heterozygous for resistance. However, Bt corn,
low-to-moderate (4, 5). The western corn rootworm (WCR), Zea mays, introduced recently does not meet this ‘‘high-dose''
Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomeli- criterion for control of western corn rootworm (WCR), Diabrotica
dae), overwinters in the egg stage in the soil. Larvae emerge in virgifera virgifera. A greenhouse method of rearing WCR on trans-
spring and begin feeding on the roots of host plants, undergoing genic corn expressing the Cry3Bb1 protein was used in which
three instars before pupating. Although rootworm-targeting Bt approximately 25% of previously unexposed larvae survived rel-
corn provides good protection of grain yield, it is common to ative to isoline survival (compared to 1– 4% in the field). After three
observe adult WCR emerging from all of the rootworm Bt generations of full larval rearing on Bt corn (Constant-exposure
products currently available. In contrast, emergence of most colony), WCR larval survival was equivalent on Bt corn and isoline
lepidopteran target pests from transgenic crops would not be corn in greenhouse trials, and the LC
expected. Beetle emergence from plots of Bt corn expressing 50 was 22-fold greater for the
Constant-exposure colony than for the Control colony in diet
Cry34Ab1⫹Cry35Ab1 proteins averaged 3.53% that of isoline bioassays with Cry3Bb1 protein on artificial diet. After six gener-
plots (6). Emergence from transgenic plots of the modified ations of greenhouse selection, the ratio of larval recovery on Bt
Cry3A protein and the Cry3Bb1 protein, which are also currently corn to isoline corn in the field was 11.7-fold greater for the
registered for rootworm control, were similar (B.E.H., V. Kaster, Constant-exposure colony than the Control colony. Removal from
H. York-Steiner, R. Kurtz, T. Clark, L. Meinke, D. Moellenbeck, selection for six generations did not decrease survival on Bt corn in
W. French, and T. Vaughn, unpublished data). Clearly, none of the greenhouse. The results suggest that rapid response to selec-
the transgenic events currently registered for WCR control tion is possible in the absence of mating with unexposed beetles,
expose larvae to a level considered high-dose. It is not known emphasizing the importance of effective refuges for resistance
what proportion of survivors of WCR-targeted Bt corn have a susceptible genotype through escaping lethal exposure to thetoxin or what proportion, if any, are genetically resistant.
Bacillus thuringiensis 兩 toxicity assay 兩 MON863 兩 reciprocal cross 兩 Here we report evolution of resistance to transgenic corn expressing the Cry3Bb1 protein within three generations ofselection under greenhouse conditions allowing relatively high larval survival (25% versus 1–4% under field conditions). Four ‘‘high-dose/refuge strategy'' is required in many areas as a colonies were subjected to different regimes of exposure to Bt means of delaying the evolution of resistance to crops corn: exposure as neonates (Neonate-exposure colony), expo- expressing transgenic insecticidal proteins derived from the soil sure as late instars (Late-exposure colony), constant exposure bacterium Bacillus thuringiensis (Bt) (Berliner) (1). This strategy throughout larval development (Constant-exposure colony), and involves an extremely high concentration of toxin (25 times the an unexposed control (Control colony). On-plant rearing con- amount needed to kill 99% of the susceptible insects) to ensure ditions differed between colonies to achieve differing Bt expo- that heterozygotes do not survive exposure in the Bt crop, thus sures (see Materials and Methods below). After three generations making resistance functionally recessive (2). In addition, a of selection, the LC nearby refuge is maintained where the pests do not encounter Bt 50 of the Constant-exposure colony was approximately 22-fold greater than the LC toxin. It is expected that a large number of susceptible pests 50 of the Control colony. After six generations, percent survival on Bt corn relative emerging from the refuge will mate with any resistant individualsemerging from the Bt field.
The duration of susceptibility of insect pests to Bt toxins Author contributions: B.D.S., T.W.S., and B.E.H. designed research; L.N.M., M.L.H., N.J.M., depends on many factors, including dose of the toxin. Although and T.A.S. performed research; M.R.E. analyzed data; and L.N.M., B.D.S., N.J.M., T.W.S., and most Bt toxins targeted toward lepidopteran pests meet the B.E.H. wrote the paper.
high-dose standard defined above, Cry1Ac targeted toward The authors declare no conflict of interest.
Heliocoverpa zea (Hu ¨bner) is not high-dose (2). This same This article is a PNAS Direct Submission.
protein does meet the high-dose standard in the context of See Commentary on page 19029.
targeting Heliothis virescens (Fabricius), a closely related He- 1To whom correspondence should be addressed. E-mail: [email protected].
liothine species often found within the same Bt cotton, Gos- This article contains supporting information online at sypium hirsutum L., fields. An increase in resistance alleles has been reported for several field populations of H. zea, but not in 2008 by The National Academy of Sciences of the USA PNAS 兩 December 9, 2008 兩 vol. 105 兩 no. 49 兩 19177–19182

(A) Mean (⫾ SEM) number of larvae from different laboratory colonies recovered during trials on Bt (black bars) and nontransgenic isoline (white bars) corn in the field after six generations of selection. (B) Percentage of survivors on Bt corn relative to survivors on isoline corn from each labora- tory colony in the field. Although untransformed data are shown, analyses were performed using square root (x ⫹ 0.5) transformed data (A) and ranktransformed data (B). Bars with the same letters are not significantly different (P ⱖ 0.05). For (A) capital letters indicate comparisons between isoline and Btwithin colonies and lowercase letters indicate comparisons between colonies within isoline or Bt corn.
exposure colonies in generations 3 and 6 Control F Constant F Constant M Control M Diet Bioassays. After three generations of selection, the resistance
ratio of the Constant-exposure colony was 22.22 After six generations of selection, the resistance ratio of the Mean (⫾ SEM) number of larvae from different laboratory colonies Constant-exposure colony was still greater than that of the Con- recovered during trials on Bt (black bars) and nontransgenic isoline (white trol and Neonate-exposure colonies, but was only 4.14. The LC50 bars) corn (A) in the greenhouse after three generations of selection and (B) of the Constant-exposure colony did not decrease, but the ratio in the greenhouse after six generations of selection. The Constant-exposurecolony after six generations of selection was further tested after (C) six was lower because of the relatively high Control colony LC50 generations of removal from selection, and (D) reciprocal crosses with the (5.75) Following removal from selection, the resis- unselected Control colony (F ⫽ female, M ⫽ male). Although untransformed tance ratio of the removal from selection colony (5.48) was data are shown, analyses were performed using square root (x ⫹ 0.5) trans- higher than that of the Constant-exposure colony (2.85). Vari- formed data. Bars with the same letters are not significantly different (P ⱖ ation in response to Cry3Bb1 was reported among geographi- 0.05). Capital letters indicate comparisons between isoline and Bt within cally distinct WCR populations tested before widespread com- colonies. Lowercase letters indicate comparisons between colonies within mercial use of Bt corn (5), providing a basis for comparison. The isoline or Bt corn.
LC50 values for the Control, Neonate-exposure, and Late-exposure colonies were in the range of those populations, but theLC50 value of the Constant-exposure colony was more than to isoline corn was 11.7-fold greater in the field for the Constant- fivefold the average LC50 value previously reported (5).
exposure colony than for the Control colony.
Field Experiment. Because selection for resistance to Bt proteins
Results and Discussion
under laboratory conditions has rarely resulted in strains capable The WCR is an adaptively dynamic insect pest with documented of surviving on Bt crops in the field (11), we evaluated all resistance to chemical controls targeting larvae (7), chemical con- colonies under field conditions. Significantly more WCR larvae trols targeting adults (8), and crop rotation commonly used as a from the Constant-exposure colony were recovered from Bt cornthan from the Control or Neonate-exposure colonies (Fig. 2A).
cultural control method (9). Interestingly, insecticide resistance in Larval survival on Bt corn relative to isoline corn in the field was this pest may persist for many generations in the absence of significantly greater for the Constant-exposure colony (44.4%) selection (10). Here, we describe the evolution of resistance to a than for the Control colony (3.79%) and the Neonate-exposure low-to-moderate dose Bt product under greenhouse conditions.
colony (1.15%), but not for the Late-exposure colony (14.4%)(Fig. 2B). The magnitude of the difference in survival on Bt Greenhouse Experiments. When WCR survivors of Bt corn were
relative to isoline between the Constant-exposure colony and the selected and mated in the laboratory, resistance evolved in as few Control colony was 11.7-fold. There were no significant differ- as three generations. In greenhouse experiments, the number of ences in the average dry weight of larvae recovered for any WCR larvae recovered from the Constant-exposure colony was treatment, implying again (12) that once WCR establish on equivalent from both Bt and isoline corn after three and six Cry3Bb1 Bt corn, growth is relatively normal, although adult generations of selection (Fig. 1 A and B). Significantly more emergence is generally delayed (6).
adults of Constant-exposure and Late-exposure colonies were Although larval survival on Bt corn and isoline corn in the recovered from Bt corn than adults of Control or Neonate- greenhouse was equivalent after three and six generations of Meihls et al.

selection (Fig. 1 A and B), significantly more WCR larvae were proteins also follow this profile (T. Vaughn, personal commu- recovered from isoline corn than from Bt corn in the field, even nication). Interestingly, behavioral responses to toxins can pre- for the Constant-exposure colony (Fig. 2 A). In attempting to vent and even decrease the levels of physiological resistance in compare greenhouse and field assays of WCR on Bt corn, it is insect populations (21), an important result suggested by previ- important to note that survival of natural field populations of ous modeling work (22).
WCR on Bt corn relative to survival on isoline corn is in therange of 1–5%, depending on which of three registered products Removal from Selection. After six generations of greenhouse selec-
are evaluated. For plants expressing Cry3Bb1, survival relative tion on Bt corn for the Constant-exposure colony and an additional to isoline was 1.34% when averaged across nine different fields six generations of removal from selection (i.e., rearing on nontrans- in Missouri, Iowa, South Dakota, and Nebraska while actual genic plants), the number of larvae recovered from Bt corn (Fig.
survival on Bt plants was only 0.042% (B.E.H., T. Clark, L.
1C) and the number of adults recovered did not differ Meinke, D. Moellenbeck, W. French, and T. Vaughn, unpub- significantly from the numbers recovered from isoline corn. Finally, lished data). Actual survival to adulthood on nontransgenic the LC50 of the Removal from selection colony remained higher plants in the field will usually vary from 1 to 10% (13) and was than that of the Control colony It should be noted that 3.1% when averaged across the same nine fields. In greenhouse the LC50 of the Constant-exposure colony and the Removal from trials reported here and additional trials we have conducted with selection colony were lower than the LC50 of the Constant-exposure Cry3Bb1 plants, survival of unselected WCR larvae on Bt corn colony from previous generations.
relative to survival on isoline was approximately 25% while Parimi, et al. (10) evaluated laboratory and field strains of actual survival was usually about 20% on isoline and 5% on Bt WCR for resistance to aldrin and methyl-parathion. As observed when infested with WCR eggs (actual survival on isoline corn in greenhouse results with the Removal from selection colony on may approach 50% when infested with neonate larvae). The Bt corn, resistance to both aldrin and methyl-parathion was current field experiment was terminated before adult emergence relatively stable in the absence of selection pressure. The chem- to prevent escape of resistant insects. Because larvae were ical class to which aldrin belongs was banned from use in 1972.
recovered rather than adults, any mortality that might have Since resistance had evolved before the 1972 ban (7), exposure occurred late in larval development or during pupation was not of WCR to aldrin has been declining or absent for more than measured, although most WCR late instar larvae survive to the thirty years, yet resistance has remained high (10). Resistance to adult stage (14, 15).
methyl-parathion was not documented until the mid-1990s (8), but resistance to this chemical also has persisted without addi- Reciprocal Crosses. Performance of progeny from reciprocal
tional selection pressure.
crosses between the Constant-exposure colony and the Controlcolony on Bt corn in the greenhouse did not differ significantly Genetic Evaluation. Neutral genetic diversity in each colony was
from the Constant-exposure colony in terms of number of larvae tracked using 11 microsatellite markers. The estimated effective recovered (Fig. 1D), average weight, and number of adults population sizes of the four colonies were comparable, with emerged The percentage of larvae recovered from Bt overlapping confidence intervals and on the order of 100 indi- relative to isoline corn was 44.3% for the Control colony, 58.8% viduals. These population sizes were sufficiently small that for the cross of Constant-exposure males by Control females, significant changes in microsatellite allele frequencies were 73.0% for the opposite reciprocal cross, and 120.2% for the observed between generations within each colony with the Constant-exposure colony. These same percentages for adult exception of the Control colony between generations 13 and 14 emergence were 45.9%, 62.0%, 48.4%, and 77.3%, respectively.
and the Constant-exposure colony between generations 8 and 9 In contrasts of the ratio of Bt survivors:isoline survivors (after six generations of exposure to Bt). Although the changes (combining larval and adult data), there was no significant in allele frequencies were significant, their magnitude was small; difference between the reciprocal crosses (F ⫽ 0.24; df ⫽ 1, 289; the largest value of FST, the proportion of genetic variation due P ⫽ 0.624), indicating that no significant maternal effects were to differences between samples, was 3.18% between the Control evident within the crosses. In the same analysis, nonrecessive colony at generation 9 and the initial F1 population used to effects were highly significant (F ⫽ 23.7; df ⫽ 1, 289; P ⫽ 0.0001), found the four colonies. However, the population sizes were not but no dominance effect was found (F ⫽ 0.23; df ⫽ 1, 289; P ⫽ so small that they caused genetic diversity to be lost from the 0.634). The dominance value (h) was 0.285 for larvae and 0.296 colonies during the course of the experiment. The mean ex- for adults. Both the linear contrasts and the calculations of h pected heterozygosity (HE) of the initial F1 between the wild point to nonrecessive inheritance of resistance.
type insects and the nondiapausing strain was 0.478. This mea- Regression of LC50 on relative survivorship (Bt :isoline) of sure of genetic diversity did not change significantly over time in larvae was significant (F ⫽ 6.08; df ⫽ 1, 9; P ⫽ 0.0390), yielding any colony, nor did it differ between the parents and F1 of the a regression equation of relative survival ⫽ 44.8226 ⫹ reciprocal crosses between the Control and the Constant- 1.199106*LC50 (r2 ⫽ 0.43). However, when examining only the exposure colonies. These results indicate that the biological reciprocal crosses, the LC50 data from diet bioassays differences observed between the colonies were due to the were characteristic of susceptible insects whereas the greenhouse selection regime imposed and not stochastic genetic processes results were characteristic of resistant insects (Fig. 1D, such as genetic drift or founder effects.
The cause for this difference remains unknown. One possiblecontributing reason for differences between on-plant and diet Research Implications. Results with the Neonate-exposure and
bioassays with reciprocal crosses could be the role of feeding Late-exposure colonies may simulate grassy weeds serving as behavior. Dramatic differences between the feeding behavior of alternate hosts near Bt corn (23) or a mixture of Bt and isoline WCR larvae on Bt corn and isoline corn suggest neonate larvae corn as has been proposed as a refuge strategy by Pioneer®.
alter feeding behavior to reduce exposure to Bt proteins (16).
Although we only have one colony per treatment, our data For low-to-moderate dose toxins, any allele that confers even suggest that selection for resistance may be minimal when slight resistance is expected to be favored by natural selection neonate larvae are exposed to Bt corn but development is (17). Genes with small effects are often common in populations completed on isoline corn (the Neonate-exposure colony was not and response to selection can be very rapid (18, 19). Root significantly different from the Control colony for any parameter growing points are higher in total soluble protein compared to of resistance). However, in a scenario where initial development older root tissue; Cry34Ab1⫹Cry35Ab1 (20) and Cry3Bb1 occurs on grassy weeds and the weeds are then sprayed with Meihls et al. PNAS 兩 December 9, 2008 兩 vol. 105 兩 no. 49 兩 19179

herbicide, or in a seed-mix scenario where isoline food resources colony was exposed to Bt corn (MON863, Monsanto Company, variety DKC are significantly depleted forcing larvae to move (24), resistance 60 –12) as neonate larvae but subsequently reared on isoline, the Late- might be expected to evolve, given that survival of the Late- exposure colony was reared on isoline corn for 1 week and then Bt corn from exposure colony on Bt corn relative to isoline survival was second instar to pupation, and the Constant-exposure colony was rearedsolely on Bt corn as larvae (except as described below).
significantly (3.8-fold) greater than the Control colony in the For the Control colony, cohorts of 125 neonate larvae of hatching eggs covered by 1 cm of soil were transferred via a fine nylon artist's brush to Recently, Lefko, et al. (20) documented an increased WCR seedling corn (approximately 45 seeds, 4 d after germination) in 15 cm ⫻ 10 cm survivorship from F1 to F9 of 15.1- and 58.5-fold for populations oval containers (708 ml, The Glad Products Company) filled approximately 4 from Rochelle, IL and York, NE, respectively, selected to survive cm deep with a growth medium of 2:1 autoclaved soil and ProMix™ (Premier on event DAS-59122–7 containing the Cry34Ab1⫹Cry35Ab1 pro- Horticulture Inc.). After 7 d, the living corn was cut at the soil surface, and the teins. Despite up to a 5,850% increase in survivorship, damage to remaining contents transferred upside down to a 33 cm ⫻ 19 cm container (5.7 DAS-59122–7 per 100 eggs from the York selected population in liters, Sterilite Corporation) with new growth medium (approximately 115 the greenhouse only increased 350% from F1 to F11 and Bt corn seeds, 4 d after germination) to allow larvae to complete development andpupate. The Neonate-exposure colony was reared identically to the Control was still significantly less damaged than isoline roots. We did not colony, but the neonate larvae were first placed on a germinated Bt corn collect damage ratings, but given similar root protection in the field seedling without soil and then the seedling plus larvae were transferred to to DAS-59122–7, we would expect damage increases to Bt corn to isoline corn. The Late-exposure colony was reared the same as the Control be similar to Lefko, et al. (20). Performance of each product in the colony for the first week, but second instar larvae were removed from their field might be better, given differing selection intensities between first container (isoline corn) using modified Tullgren funnels and then trans- the greenhouse and field, but this was not tested in either study.
ferred to a 15.5 liter pot with 2 Bt corn plants at approximately V6-V7 (28).
Lefko, et al. (20) did not evaluate their selected populations in the Late-exposure colony larvae finished their development on Bt corn plants and field, and we did not evaluate plant damage in the field or pupated in the pots. Just before predicted adult emergence, one plant was cut greenhouse, so direct comparison of field survivorship is not at the base and the other corn plant was passed through a hole in insect netting, which was secured around the corn plant stalk with a cable tie and tothe pot with a rubber band. Finally, the Constant-exposure colony was reared Although we have not included summaries of colony perfor- exclusively on Bt corn plants (except as described below). This involved large mance from generation to generation as in Lefko, et al., we do beds (1.2 m wide ⫻ 7.5 m long ⫻ 25 cm deep) of the same growth medium used have similar data. For the Constant-exposure colony, 1.5% of above in which 294 kernels of Bt corn were planted. Each plant was infested eggs survived Bt corn the first generation (F1) of rearing in the with 200 eggs at approximately V3 stage during the first few generations with greenhouse. After six generations of selection on Bt corn, ⬇4.1% egg hatch at approximately V5– 6. The number of eggs per plant was reduced of the eggs survived Bt corn to produce adults in greenhouse in later generations to 100 with infestation at V1–2 and egg hatch approxi- rearing. In a controlled greenhouse experiment, an average of mately V4. Beds were covered with fine mesh screen to prevent adult escape 2.73 adults were produced per plant on Bt corn from 50 5– 6 weeks following infestation, depending on temperature. Adults from all Constant-exposure eggs from generation 6 Thus, colonies were collected daily.
To ensure enough individuals to maintain the colony as well as conduct adult production on Bt corn increased from 1.5% for F1 to 5.5% controlled greenhouse and field experiments in which eggs were removed for F6, an increase of about 3.7-fold in six generations of from the colony, it was sometimes necessary to rear one generation on isoline selection. One reason that the increase in survival to the adult corn before initiating another generation of selection on Bt. Thus, ‘‘genera- stage may not have increased as rapidly in the current study as tion 6'' of the Constant-exposure colony refers to six generations of selection the 58.5-fold increase found in Lefko, et al. (20) is that relative on Bt, but these generations were interspersed with three additional gener- to isoline survival, we already had a high rate of survival on Bt ations of increase on isoline corn. When comparing colonies, actual genera- corn under our greenhouse rearing conditions (approximately tion numbers were not the same for all colonies. The Figs., Table, and text refer 25% relative to isoline compared to 1–4% relative to isoline to generations of selection, not total generations in culture. For the Constant- survival in the field).
exposure colony, after generation 2, rather than putting eggs back onto Bt, alleggs were put onto isoline to increase the population size of the colony.
Taken together, our results suggest that rapid response to Generations 4 and 5 were also increased on isoline, primarily in preparation selection is possible in the absence of mating with unexposed for the field trial, which needed a large number of eggs that would not go beetles. These data emphasize the importance of effective back into the colony. The Neonate-exposure colony was not increased on refuges for resistance management, especially for low-to- isoline except in advance of the field experiment. The Late-exposure colony moderate dose toxins.
was increased after generation 3 and generation 5 on isoline corn.
Materials and Methods
Greenhouse Experiments. Standard procedures. WCR survival was evaluated on
Colony Development. Eggs from a feral WCR population collected near Dodge
Bt (DKC 60 –12) and isoline (DKC 60 –15) corn in greenhouse trials. For each City, KS, in July 2002 by French Agricultural Research were purchased and used replication of each treatment, three pots were planted with two corn seeds for an unrelated field experiment in Missouri in 2003. Beetles from the 2003 each; two pots (3.8 liters) for larval recovery and one pot (19 liters) for adult experiment were collected from susceptible corn, kept alive, and brought to recovery. Following germination, seedlings were thinned to one plant per pot.
the laboratory where they were mated with each other and resulting eggs The same growth medium was used as for rearing. To prevent larval escape overwintered at 8 °C. In April 2004, eggs were removed from cold storage, (23), drainage holes on all pots were fitted with 114-␮m stainless steel mesh reared on isoline corn, and resulting adults were crossed reciprocally with a (TWP Inc.). Plants were watered as needed and fertilized approximately 6 wk nondiapausing WCR strain (25) so that generation time could be reduced from after planting with 1.25 ml of Peters Professional® Multi Purpose 20 –20-20 9 months (1 year in the field) to 2 months. The wild-type genes were intro- (The Scotts Company LLC).
gressed because the nondiapausing colony has been maintained in the labo- Three weeks after planting, pots were infested with 50 WCR eggs sus- ratory for more than 200 generations and has lost genetic variability (26).
pended in 0.15% agar solution pipetted into a 2.5-cm hole in the soil. Holes After combining eggs from the two reciprocal crosses, a total of 4,242 adults were covered and the plants lightly watered. At infestation, a subsample of emerged that laid a total of 241,000 eggs. From these eggs, four separate eggs was placed on moist filter paper in a Petri dish. The dish was placed near colonies were established, each fed optimally as adults but differing in larval the pots and monitored for percent hatch and time to hatch.
diet. Adults were held in the laboratory under 14:10 [L:D] photoperiod and Larvae were recovered from two sets of pots 1 and 2 wk following peak egg 25 °C. Adults from all colonies were maintained in 30 ⫻ 30 ⫻ 30 cm cages hatch. Recovery was accomplished by cutting plants near the soil surface, then (MegaView) and provided with artificial diet (27), fresh non-Bt corn leaves, emptying the pots into modified Tullgren funnels equipped with a 60 W light and water. Oviposition substrate consisted of 1 cm moist 70 mesh (212 ␮m) bulb. The root ball was carefully broken to encourage drying. Larvae were sieved soil in Petri dishes with the surface scarified to promote oviposition and collected in attached pint jars filled with 2.5 cm water, and were subsequently dishes were replaced weekly (twice weekly for the first year). Eggs were transferred after 2 and 4 d to 95% ethanol. Larval dry weight was obtained recovered by rinsing the soil through a 60 mesh sieve (250 ␮m) with water. The after desiccation in an oven (Thelco model 16, GCA/Precision Scientific Co.).
Control colony was reared on isoline corn (DKC 60 –15), the Neonate-exposure The corn plant in adult emergence pots was passed through a hole in insect Meihls et al.

netting, which was secured around the stalk with a cable tie and to the pot generation used to initiate the four colonies and at various generations with a rubber band. Pots were checked for adults three times weekly until no thereafter, as well as the parents and F1 generations of the reciprocal crosses adults were collected for two consecutive weeks. Recovered adults were between the Control and Constant-exposure colonies. Samples of the colonies stored in 95% ethanol until they could be sexed, counted, and dry weight were examined at the following generations: Control at generations 4, 10, 13, taken as described for larvae. Greenhouse air temperature was recorded on an and 14; Neonate-exposure at generations 4 and 9; Late-exposure at genera- hourly basis (HOBO, model H08 – 001-02).
tions 3 and 6; and Constant-exposure at approximately generations 8 and 9 (six Generations 3 and 6. All four colonies were evaluated after generations 3
generations of selection).
(August 2005) and 6 (June 2006) of selection of the Constant-exposure colony DNA was extracted from adult beetles using AquaPure Genomic DNA kits using the standard procedures described above. During the two experiments, (Bio-Rad). The microsatellite loci were amplified by PCR in three multiplex hourly air temperatures in the greenhouse averaged 23.5 ⫾ 0.09 °C SE (range reactions using multiplex PCR kits (Qiagen) according to the manufacturer's 12.6 –33.6 °C) and 26.4 ⫾ 0.08 °C SE (range 18.3– 42 °C), respectively. Soil instructions in a 10 ␮l volume with 20 ng genomic DNA. One of the PCR primers temperatures likely did not vary as extensively. The larval recovery experiment for each microsatellite was labeled with a fluorescent dye that allowed the was designed as a randomized complete block split-plot with the main plot amplicons to be detected and sized using a Beckman-Coulter CEQ 8000 being treatment and the subplot being recovery date. The adult recovery genetic analysis system (Beckman-Coulter). The number of individuals success- experiment was designed as a randomized complete block. There were at least fully analyzed from each sample ranged from 26 to 60.
15 replications for each larval recovery time and adult emergence (25 repli-cations for adult emergence at generation 3).
Statistical Analysis. Greenhouse experiments. Although nontransformed data
Reciprocal Crosses. Newly emerged adults from the Control (generation 13) and
are shown in the figures, data from all experiments were square root (x ⫹ 0.5) Constant-exposure colonies (generations 6 and 7 of Bt exposure) were placed transformed before analysis to meet the assumptions of the analysis (31).
in separate rearing cages (30 ⫻ 30 ⫻ 30 cm) (MegaView). Males were segre- Larval recovery data were analyzed as a randomized complete block three way gated for 10 days to reach sexual maturity before introduction to females. At factorial design (four colonies, two corn types, and two larval recovery times) least 100 virgin females from the Control colony were allowed to mate with using PROC MIXED of the SAS statistical package (32). The model contained males from the Constant-exposure colony and vice versa. Adults were main- the main effect of colony, corn type, larval recovery date, and all possible tained as described above.
interactions. Replications were included as the random variable. A separate Offspring of the reciprocal crosses, along with the Control and Constant- analysis was done for number of larvae recovered and average larval weight.
exposure colonies, were evaluated for growth and survival under standard Adult emergence data were analyzed separately as a randomized complete greenhouse procedures (December 2006). Hourly air temperature averaged block design using PROC MIXED. Since there was no interaction of colony ⫻ 22.9 ⫾ 0.07 °C SE (range 9 –38.3 °C). The larval recovery experiment was collection period (larval sample 1 and 2), the main effect of colony is presented designed as a randomized complete block split-plot with the main plot being in Figs. 1–2.
treatment and the subplot being larval recovery date with 20 replications. The Reciprocal crosses were further analyzed to specifically test for maternal, adult recovery experiment was designed as a randomized complete block with nonrecessive, and dominance effects. For each replication of each collection 20 replications.
period (1st larval, 2nd larval, and adult), the number of individuals recovered Removal from Selection. A subset of the Constant-exposure colony was removed
from Bt corn was divided by the number of individuals recovered from isoline from selection after six generations on Bt corn and reared on isoline for six corn to provide the colony's relative survival on Bt corn, adding 1 to the generations. Larvae were evaluated for growth and survival using the stan- numerator and denominator to avoid division by zero. Because model as- dard greenhouse procedures described above along with larvae from the sumptions were not initially met, ratios were log transformed (31). Data were Control colony and the Constant-exposure colony (July 2007). Greenhouse air analyzed as a randomized complete block design using PROC MIXED. Since temperatures during this experiment averaged 27.7 ⫾ 0.31 °C SE (range there was no interaction of colony ⫻ collection period (larval sample 1, 2, and 17.1–39.7 °C). The larval recovery experiment was designed as a randomized adult emergence), the main effect of colony is presented. Specific contrasts complete block split-plot, with the main plot being treatment and the subplot were made between the two reciprocal crosses to test for maternal effects.
being larval recovery date with 15 replications. The adult recovery experiment Since they were not significant, these were pooled in the contrasts that follow.
was designed as a randomized complete block with 15 replications.
Contrasts between the Control colony and the Constant-exposure colony wereused to test for nonrecessive effects. Dominance effects were evaluated by Field Experiment. All colonies were evaluated on both Bt (variety DKC 60 –12)
contrasting the parental colonies and their reciprocal crosses. In addition, the and isoline (variety DKC 60 –15) corn in field experiments at the Bradford dominance value (h) was calculated from the reciprocal cross larval recovery Research and Extension Center of the University of Missouri near Columbia, and adult recovery data, as suggested by Tabashnik, et al. (3). Dominance MO in 2006. The experiment was designed as a randomized complete block values of 0 indicate completely recessive resistance, while dominance values of with ten replications. Each replicate of each treatment consisted of a single 1 indicate completely dominant resistance.
plant infested with 500 viable eggs from one of the above colonies. To ensure Field experiment. The number of larvae recovered in the field (Fig. 2 A) were
adequate numbers of eggs, each colony was increased on isoline in time to lay analyzed as a randomized complete block design using PROC MIXED. The model eggs for the field experiment. Each infested plant was destructively sampled contained the main effect of colony, corn type, and all possible interactions. In by putting the whole root ball with soil in an onion bag, which was then hung addition, within each replication, the ratio of larvae recovered from Bt corn:iso- in a greenhouse with the cooling system turned off. Temperatures in such a line corn was analyzed because the ratio represents relative survival on Bt corn greenhouse in late June in Missouri are often 50 – 65 °C. Under these condi- and it controls for differences in egg hatch between strains (Fig. 2B). Because tions, larvae leave the hot and drying soil in search of a more suitable model assumptions were not met initially, the ratios were rank transformed (33) environment (12, 24). Larvae were captured in water pans below each root and analyzed as a randomized complete block design using PROC MIXED; how- ball, and were transferred to 95% ethanol at least twice daily. Natural infes- ever, untransformed data averaged across replications are presented. Only bio- tation by the southern corn rootworm, D. undecimpunctata howardi Barber, assays in which control mortality was ⬍20% and which had at least three is possible in central Missouri, so the species of each rootworm larva was concentrations producing mortality ⬎0 and ⬍100% were subjected to determined based on the presence or absence of urogomphi on the posterior further statistical analysis. Bioassays were conducted in duplicate on three margin of the anal plate (29). Most, but not all, southern corn rootworm larvae different dates, depending on availability of eggs. Mortality data were can be detected by this technique (12). The number of WCR larvae recovered analyzed by probit analysis using POLO-PC (34). Resistance ratios were and larval dry weight were recorded.
calculated by dividing the LC50 of the selected colony by the LC50 of theaccompanying Control colony.
Diet Bioassays. Bioassays were conducted by exposing neonate larvae to increas-
A regression analysis was performed to determine the ability of LC50 data ing concentrations of Cry3Bb1 applied to artificial diet. The colonies were tested from diet bioassays to predict relative survival of insects on plants. Larval LC50 at generations 3 (July 2005) and 6 (June 2006). Offspring of reciprocal crosses data were collected from each colony at generations 3 and 6 and from larvae (June and November 2006) and the colony removed from selection (August 2007) of the reciprocal crosses. These data were paired with their respective relative were also evaluated, along with the Control and Constant-exposure colonies.
survivals (number of individuals recovered from Bt corn/number of individuals Each generation was increased on isoline corn before diet bioassay evaluations to recovered from isoline corn) for each colony at each generation. The regres- separate genetic effects from other Cry3Bb1 effects. All bioassays were con- sion was performed using PROC REG of the SAS statistical package.
ducted as described by Siegfried, et al. (5).
Genetic evaluation. Genepop 4.0 (35) was used to calculate expected heterozy-
gosities (HE) as a measure of overall genetic diversity for each sample, FST which
Genetic Evaluation. Changes in genetic diversity of the colonies over time were
is the proportion of genetic variation due to differences between samples and tracked using 11 microsatellite loci (30). Samples were examined from the F1 a measure of allele frequency differences, and to perform exact tests of allele Meihls et al. PNAS 兩 December 9, 2008 兩 vol. 105 兩 no. 49 兩 19181

frequency difference between samples. Differences in HE between samples anonymous reviewers for valuable suggestions on earlier drafts. Aaron were tested using a Wilcoxon test for matched pairs. The effective population Gassmann also assisted in calculations of maternal, nonrecessive, and size of each colony was estimated with the pseudolikelihood method (36).
dominance effects as well as the dominance value, h. This research wassupported, in part, by USDA-ARS, Biotechnology Risk Assessment Award ACKNOWLEDGMENTS. We thank R. Bukowsky and J. Barry for technical
No. 2006 –33522-17716, the University of Missouri Division of Plant Sci- assistance. We thank Fred Gould, Aaron Gassmann, Tom Coudron, and two ences, and by Monsanto Corporation.
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Meihls et al.

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