Increased survival of western corn rootworm on transgenic corn within three generations of on-plant greenhouse selection

Greenhouse Experiments.

When WCR survivors of Bt corn were selected and mated in the laboratory, resistance evolved in as few as three generations. In greenhouse experiments, the number of WCR larvae recovered from the Constant-exposure colony was equivalent from both Bt and isoline corn after three and six generations of selection (Fig. 1 A and B). Significantly more adults of Constant-exposure and Late-exposure colonies were recovered from Bt corn than adults of Control or Neonate-exposure colonies in generations 3 and 6 (supporting information (SI) Fig. S1 A and B).

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Fig. 1.

Mean (± SEM) number of larvae from different laboratory colonies recovered during trials on Bt (black bars) and nontransgenic isoline (white bars) corn (A) in the greenhouse after three generations of selection and (B) in the greenhouse after six generations of selection. The Constant-exposure colony after six generations of selection was further tested after (C) six generations of removal from selection, and (D) reciprocal crosses with the unselected Control colony (F = female, M = male). Although untransformed data are shown, analyses were performed using square root (x + 0.5) transformed data. Bars with the same letters are not significantly different (P ≥ 0.05). Capital letters indicate comparisons between isoline and Bt within colonies. Lowercase letters indicate comparisons between colonies within isoline or Bt corn.

Diet Bioassays.

After three generations of selection, the resistance ratio of the Constant-exposure colony was 22.22 (Table S1). After six generations of selection, the resistance ratio of the Constant-exposure colony was still greater than that of the Control and Neonate-exposure colonies, but was only 4.14. The LC₅₀ of the Constant-exposure colony did not decrease, but the ratio was lower because of the relatively high Control colony LC₅₀ (5.75) (Table S1). Following removal from selection, the resistance ratio of the removal from selection colony (5.48) was higher than that of the Constant-exposure colony (2.85). Variation in response to Cry3Bb1 was reported among geographically distinct WCR populations tested before widespread commercial use of Bt corn (5), providing a basis for comparison. The LC₅₀ values for the Control, Neonate-exposure, and Late-exposure colonies were in the range of those populations, but the LC₅₀ value of the Constant-exposure colony was more than fivefold the average LC₅₀ value previously reported (5).

Field Experiment.

Because selection for resistance to Bt proteins under laboratory conditions has rarely resulted in strains capable of surviving on Bt crops in the field (11), we evaluated all colonies under field conditions. Significantly more WCR larvae from the Constant-exposure colony were recovered from Bt corn than from the Control or Neonate-exposure colonies (Fig. 2A). Larval survival on Bt corn relative to isoline corn in the field was significantly greater for the Constant-exposure colony (44.4%) than for the Control colony (3.79%) and the Neonate-exposure colony (1.15%), but not for the Late-exposure colony (14.4%) (Fig. 2B). The magnitude of the difference in survival on Bt relative to isoline between the Constant-exposure colony and the Control colony was 11.7-fold. There were no significant differences in the average dry weight of larvae recovered for any treatment, implying again (12) that once WCR establish on Cry3Bb1 Bt corn, growth is relatively normal, although adult emergence is generally delayed (6).

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Fig. 2.

(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 laboratory colony in the field. Although untransformed data are shown, analyses were performed using square root (x + 0.5) transformed data (A) and rank transformed data (B). Bars with the same letters are not significantly different (P ≥ 0.05). For (A) capital letters indicate comparisons between isoline and Bt within colonies and lowercase letters indicate comparisons between colonies within isoline or Bt corn.

Although larval survival on Bt corn and isoline corn in the greenhouse was equivalent after three and six generations of selection (Fig. 1 A and B), significantly more WCR larvae were recovered from isoline corn than from Bt corn in the field, even for the Constant-exposure colony (Fig. 2A). In attempting to compare greenhouse and field assays of WCR on Bt corn, it is important to note that survival of natural field populations of WCR on Bt corn relative to survival on isoline corn is in the range of 1–5%, depending on which of three registered products are evaluated. For plants expressing Cry3Bb1, survival relative to isoline was 1.34% when averaged across nine different fields in Missouri, Iowa, South Dakota, and Nebraska while actual survival on Bt plants was only 0.042% (B.E.H., T. Clark, L. Meinke, D. Moellenbeck, W. French, and T. Vaughn, unpublished data). Actual survival to adulthood on nontransgenic plants in the field will usually vary from 1 to 10% (13) and was 3.1% when averaged across the same nine fields. In greenhouse trials reported here and additional trials we have conducted with Cry3Bb1 plants, survival of unselected WCR larvae on Bt corn relative to survival on isoline was approximately 25% while actual survival was usually about 20% on isoline and 5% on Bt when infested with WCR eggs (actual survival on isoline corn may approach 50% when infested with neonate larvae). The current field experiment was terminated before adult emergence to prevent escape of resistant insects. Because larvae were recovered rather than adults, any mortality that might have occurred late in larval development or during pupation was not measured, although most WCR late instar larvae survive to the adult stage (14, 15).

Reciprocal Crosses.

Performance of progeny from reciprocal crosses between the Constant-exposure colony and the Control colony on Bt corn in the greenhouse did not differ significantly from the Constant-exposure colony in terms of number of larvae recovered (Fig. 1D), average weight, and number of adults emerged (Fig. S1D). The percentage of larvae recovered from Bt relative to isoline corn was 44.3% for the Control colony, 58.8% for the cross of Constant-exposure males by Control females, 73.0% for the opposite reciprocal cross, and 120.2% for the Constant-exposure colony. These same percentages for adult emergence were 45.9%, 62.0%, 48.4%, and 77.3%, respectively.

In contrasts of the ratio of Bt survivors:isoline survivors (combining larval and adult data), there was no significant difference between the reciprocal crosses (F = 0.24; df = 1, 289; P = 0.624), indicating that no significant maternal effects were evident within the crosses. In the same analysis, nonrecessive effects were highly significant (F = 23.7; df = 1, 289; P = 0.0001), but no dominance effect was found (F = 0.23; df = 1, 289; P = 0.634). The dominance value (h) was 0.285 for larvae and 0.296 for adults. Both the linear contrasts and the calculations of h point to nonrecessive inheritance of resistance.

Regression of LC₅₀ on relative survivorship (Bt :isoline) of larvae was significant (F = 6.08; df = 1, 9; P = 0.0390), yielding a regression equation of relative survival = 44.8226 + 1.199106*LC₅₀ (r² = 0.43). However, when examining only the reciprocal crosses, the LC₅₀ data from diet bioassays (Table S1) were characteristic of susceptible insects whereas the greenhouse results were characteristic of resistant insects (Fig. 1D, Fig. S1D). The cause for this difference remains unknown. One possible contributing reason for differences between on-plant and diet bioassays with reciprocal crosses could be the role of feeding behavior. Dramatic differences between the feeding behavior of WCR larvae on Bt corn and isoline corn suggest neonate larvae alter feeding behavior to reduce exposure to Bt proteins (16). For low-to-moderate dose toxins, any allele that confers even slight resistance is expected to be favored by natural selection (17). Genes with small effects are often common in populations and response to selection can be very rapid (18, 19). Root growing points are higher in total soluble protein compared to older root tissue; Cry34Ab1+Cry35Ab1 (20) and Cry3Bb1 proteins also follow this profile (T. Vaughn, personal communication). Interestingly, behavioral responses to toxins can prevent and even decrease the levels of physiological resistance in insect populations (21), an important result suggested by previous modeling work (22).

Removal from Selection.

After six generations of greenhouse selection on Bt corn for the Constant-exposure colony and an additional six generations of removal from selection (i.e., rearing on nontransgenic plants), the number of larvae recovered from Bt corn (Fig. 1C) and the number of adults recovered (Fig. S1C) did not differ significantly from the numbers recovered from isoline corn. Finally, the LC₅₀ of the Removal from selection colony remained higher than that of the Control colony (Table S1). It should be noted that the LC₅₀ of the Constant-exposure colony and the Removal from selection colony were lower than the LC₅₀ of the Constant-exposure colony from previous generations.

Parimi, et al. (10) evaluated laboratory and field strains of WCR for resistance to aldrin and methyl-parathion. As observed in greenhouse results with the Removal from selection colony on Bt corn, resistance to both aldrin and methyl-parathion was relatively stable in the absence of selection pressure. The chemical class to which aldrin belongs was banned from use in 1972. Since resistance had evolved before the 1972 ban (7), exposure of WCR to aldrin has been declining or absent for more than thirty years, yet resistance has remained high (10). Resistance to methyl-parathion was not documented until the mid-1990s (8), but resistance to this chemical also has persisted without additional selection pressure.

Genetic Evaluation.

Neutral genetic diversity in each colony was tracked using 11 microsatellite markers. The estimated effective population sizes of the four colonies were comparable, with overlapping confidence intervals and on the order of 100 individuals. These population sizes were sufficiently small that significant changes in microsatellite allele frequencies were observed between generations within each colony with the exception of the Control colony between generations 13 and 14 and the Constant-exposure colony between generations 8 and 9 (after six generations of exposure to Bt). Although the changes in allele frequencies were significant, their magnitude was small; the largest value of F_ST, the proportion of genetic variation due to differences between samples, was 3.18% between the Control colony at generation 9 and the initial F1 population used to found the four colonies. However, the population sizes were not so small that they caused genetic diversity to be lost from the colonies during the course of the experiment. The mean expected heterozygosity (H_E) of the initial F1 between the wild type insects and the nondiapausing strain was 0.478. This measure of genetic diversity did not change significantly over time in any colony, nor did it differ between the parents and F1 of the reciprocal crosses between the Control and the Constant-exposure colonies. These results indicate that the biological differences observed between the colonies were due to the selection regime imposed and not stochastic genetic processes such as genetic drift or founder effects.

Research Implications.

Results with the Neonate-exposure and Late-exposure colonies may simulate grassy weeds serving as alternate hosts near Bt corn (23) or a mixture of Bt and isoline corn as has been proposed as a refuge strategy by Pioneer®. Although we only have one colony per treatment, our data suggest that selection for resistance may be minimal when neonate larvae are exposed to Bt corn but development is completed on isoline corn (the Neonate-exposure colony was not significantly different from the Control colony for any parameter of resistance). However, in a scenario where initial development occurs on grassy weeds and the weeds are then sprayed with herbicide, or in a seed-mix scenario where isoline food resources are significantly depleted forcing larvae to move (24), resistance might be expected to evolve, given that survival of the Late-exposure colony on Bt corn relative to isoline survival was significantly (3.8-fold) greater than the Control colony in the field.

Recently, Lefko, et al. (20) documented an increased WCR survivorship from F1 to F9 of 15.1- and 58.5-fold for populations from Rochelle, IL and York, NE, respectively, selected to survive on event DAS-59122–7 containing the Cry34Ab1+Cry35Ab1 proteins. Despite up to a 5,850% increase in survivorship, damage to DAS-59122–7 per 100 eggs from the York selected population in the greenhouse only increased 350% from F1 to F11 and Bt corn was still significantly less damaged than isoline roots. We did not collect damage ratings, but given similar root protection in the field to DAS-59122–7, we would expect damage increases to Bt corn to be similar to Lefko, et al. (20). Performance of each product in the field might be better, given differing selection intensities between the greenhouse and field, but this was not tested in either study. Lefko, et al. (20) did not evaluate their selected populations in the field, and we did not evaluate plant damage in the field or greenhouse, so direct comparison of field survivorship is not possible.

Although we have not included summaries of colony performance from generation to generation as in Lefko, et al., we do have similar data. For the Constant-exposure colony, 1.5% of eggs survived Bt corn the first generation (F1) of rearing in the greenhouse. After six generations of selection on Bt corn, ≈4.1% of the eggs survived Bt corn to produce adults in greenhouse rearing. In a controlled greenhouse experiment, an average of 2.73 adults were produced per plant on Bt corn from 50 Constant-exposure eggs from generation 6 (Fig. S1B). Thus, adult production on Bt corn increased from 1.5% for F1 to 5.5% for F6, an increase of about 3.7-fold in six generations of selection. One reason that the increase in survival to the adult stage may not have increased as rapidly in the current study as the 58.5-fold increase found in Lefko, et al. (20) is that relative to isoline survival, we already had a high rate of survival on Bt corn under our greenhouse rearing conditions (approximately 25% relative to isoline compared to 1–4% relative to isoline survival in the field).

Taken together, our results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles. These data emphasize the importance of effective refuges for resistance management, especially for low-to-moderate dose toxins.