Negative cross-resistance between structurally different Bacillus thuringiensis toxins may favor resistance management of soybean looper in transgenic Bt cultivars

High adoption rates of single-gene Bacillus thuringiensis (Bt) Cry1Ac soybean impose selection pressure for resistance in the soybean looper, Chrysodeixis includens, a major defoliator in soybean and cotton crops. To anticipate and characterize resistance profiles that can evolve, soybean looper larvae collected from field crops in Brazil in 2013 were selected for resistance to Cry1Ac. Using two methods of selection viz., chronic exposure to Cry1Ac cotton leaves and the seven-day larval exposure to purified Cry1Ac on the artificial diet, 31 and 127-fold resistance was obtained in 11 and 6 generations of selection, respectively. The resistance trait had realized heritability of 0.66 and 0.72, respectively, indicating that most of the phenotypic variation in Cry1Ac susceptibility of the soybean looper larvae was due to additive genetic variation. The Cry1Ac-selected populations showed positive cross-resistance to Cry1Ab (6.7–8.7 fold), likely because these Bt toxins have a very similar molecular structure. Importantly, the Cry1Ac-selected populations became more susceptible to Cry2Aa and Cry1Fa, showing negative cross-resistance (up to 6-fold, P < 0.05). These results indicate that Cry1Ac, Cry1Fa, and Cry2A are compatible in a multi-toxin approach to minimize the risk of rapid adaptation of the soybean looper to Bt toxins.


Results
Response to selection for Cry1Ac resistance. Estimates of median lethal concentration (LC 50 ) values and survival rates over the generations of selection (Fig. 1) show that soybean looper larvae increased the resistance to Cry1Ac when exposed to the Bt cotton leaf tissues throughout larval development, or to the toxin on the surface of the artificial diet for seven days of exposure. Selection with Cry1Ac overlaid on the surface of the diet resulted in higher resistance in each generation of selection (Fig. 1a), as it imposed a greater intensity of selection than did the Bt cotton leaf tissues producing Cry1Ac ( respectively). In fact, with only two generations of selection using the purified toxin, the increase in the LC 50 values was similar to that achieved in six generations of selection using the Bt cotton leaves (Fig. 1a).
Realized heritability. Realized  Level of resistance and cross-resistance. As indicated by the ratios between the LC 50 values for the selected and unselected larvae (Table 2), selection with Cry1Ac cotton leaves produced a 31-fold resistance in 11 generations of selection, while exposure to purified Cry1Ac produced a 127-fold resistance to Cry1Ac in only 6 generations of selection (Table 2). Cry1Ab LC 50 values increased 7-9 fold in both selected insect populations, indicating positive cross-resistance between Cry1Ab and Cry1Ac in the soybean looper (Table 2). In contrast, for larvae selected with Cry1Ac cotton leaves, there was a significant reduction in Cry2Aa and Cry1Fa LC 50 values of 4 and 6-fold, respectively (P < 0.05, based on the likelihood ratio test and non-overlapping fiducial limits); likewise, there was a 6-fold decrease in Cry2Aa LC 50 value for the population selected with purified Cry1Ac toxin.
These results indicate negative cross-resistance between Cry1Ac and Cry2Aa or Cry1Fa in the soybean looper, even if a conservartive criterion is used (i.e., lack of overlap between fiducial limits of the LC 50 values for unselected and selected strains) ( Table 2).
Survival assays on soybean leaf tissue. Despite the 127-fold resistance to Cry1Ac in the 1Ac-Sel soybean looper population, their larvae did not survive on Cry1Ac soybean foliage (Fig. 2). The was no significant difference in larval survival of the three insect populations on either non-Bt or Bt soybean (F 2, 110 = 1.37, P = 0.32; F 2, 109 = 0.01, P = 0.99). However, overall insect survival differed significantly between Bt and non-Bt soybean (F 1, 110 = 13545.8, P < 0.01) (Fig. 2). Most larvae of any selected or unselected populations died in 3 days on Bt soybean. On non-Bt soybean, the survival rates were similar for both selected and unselected control populations ( Fig. 2), indicating no fitness cost of resistance to early-instar larval survival.

Discussion
Field-derived larvae of the soybean looper responded to selection for resistance to Cry1Ac in the laboratory, generating up to 127-fold resistance to the toxin, which is consistent with other selection experiments using purified Bt toxin 30,[45][46][47][48] and plant leaf tissue producing Bt toxin 7,31,38,39,49 . These results indicate that either method of selection (i.e., leaf tissues of Bt plants or larval diet containing Bt toxin) can be used to produce resistant insect populations, which are important tools for anticipating the risk assessment of resistance development in field settings 7,30,47 . Whereas using purified Bt toxin on the larval diet may allow better control of the intensity of selection and avoid confounding effects of plant allelochemicals, using the Bt plant foliage may better represent the larval exposure under field settings 50 , selecting for a more realistic resistance profile. Gossypol interaction with Cry1Ac in Bt cotton may have affected the ability of soybean looper to evolve resistance as fast as with pure Cry1Ac 51 . In addition, Cry1Ac protein levels may have varied in the cotton foliage 52,53 , even though we meticulously grew cotton plants in optimized soil conditions and standardized the growth stage and the leaf age used in the experiment. To our knowledge, this is the first report of selection of Bt resistant populations of soybean looper. Importantly, despite the 127-fold resistance to Cry1Ac in the 1Ac-Sel soybean looper population, their larvae did not survive on Cry1Ac soybean foliage. The low survival of 1Ac-Sel larvae on Cry1Ac soybean foliage may be due to the relatively high titer of Bt toxin 10,54,55 or its synergism with soybean allelochemicals 56 .
The higher level of resistance reached by the 1Ac-Sel population indicates that the rate of resistance evolution to Cry1Ac in soybean looper is linked to the intensity of the selection 57 . The realized heritability (h 2 ) values for Cry1Ac resistance were quite high (0.66 and 0.72), which means that most of the phenotypic variation for Cry1Ac resistance in the selected soybean looper populations is due to genetic variation 50 , and indicates that the soybean looper has high potential to develop resistance to Cry1Ac. The higher heritability value for the selection using  Table 1. Estimation of realized heritability (h 2 ) and number of generations to a 10-fold increase in resistance to the Cry1Ac Bacillus thuringiensis toxin in two populations of soybean looper selected using chronic exposure to Cry1Ac Bt cotton leaves (BG1-Sel) or seven-day larval exposure to purified Cry1Ac on the artificial diet (1Ac-Sel). LC 50 , lethal concentration of Cry1Ac needed to kill 50% of the larvae exposed to the toxin. a Intensity of selection was calculated according to Tabashnik  Cry1Ac on artificial diet is indicative of a greater contribution of genes as compared to that for selection using Bt cotton leaves. The latter reflects that low concentration of Cry1Ac and presence of allelochemicals. The slow resistance evolution in the soybean looper using Bt cotton may reflect a likely scenario for field-evolved resistance in the near future in a soybean agroecosystem, particularly if Bt soybean cultivars carry natural resistance to the looper and/or expresses high Bt toxin content. Despite trying to obtain resistant strains representative of those found in the field by mimicking field conditions in our laboratory selection experiment 50,57 we cannot guarantee that the resistance is exactly like that which may evolve in the field 58 , because the conditions will differ under field settings. Nevertheless, laboratory selection experiments have often produced resistance similar to field-evolved resistance 8 Table 2. Resistance and cross-resistance to Bacillus thuringiensis (Bt) toxins in two soybean looper populations, one selected using chronic exposure to Cry1Ac Bt cotton leaves (BG1-Sel) and the other using seven-day larval exposure to purified Cry1Ac on the artificial diet (1Ac-Sel). The bioassays were conducted during the last generation of selection. a LC 50 , (Lethal Concentration causing 50% mortality, in ng/cm 2 ) was estimated by probit analysis using Polo-Plus 79 . b Resistance ratio = LC 50 selected population/LC 50 for control population, indicates the level of resistance or cross-resistance, that is, how many times the selected population is less susceptible than the control, unselected population to a particular toxin; values in parentheses represent the 95% confidence limits for the resistance ratio 79 . c Chi-square statistic with its P value for df = 5. d Number of insects tested in the bioassays.  that the pre-existing Cry1Ac resistance alleles 12 may not be so rare in the field (i.e., >0.001), such that the risk of populations of soybean looper to evolve field resistance to Cry1Ac soybean should be further investigated. Selection for Cry1Ac resistance resulted in different levels of cross-resistance to other Cry toxins regardless of the method of toxin exposure. Importantly, our estimated LC 50 values are comparable to those reported for soybean loopers in Brazil 60 and the United States 61 . This latter study also reported that Cry1Ac and Cry1Fa toxins have specific binding sites on the midgut brush border membrane vesicles of soybean looper larvae, although the toxins share some, but not all, binding sites in the insect midgut 61 . Here, Cry1Ac and Cry1Ab showed low but significant (i.e., 6.7-8.7 fold) positive cross-resistance, which is not surprising given the likely resistance mechanism 62 and the similarity in the amino acid sequences of domains II and III (i.e., 99% and 51%, respectively) 32 . Despite a paucity of published reports on competition binding assays between Cry1Ac and Cry1Ab in the soybean looper, these toxins do share binding sites 63,64 or show positive cross-resistance 33,65 in other Lepidoptera.
In addition, there was no positive cross-resistance between Cry1Ac and Cry1Fa or Cry2Aa in the two selected soybean looper populations, supporting an absence of common binding sites for these pairs of toxins on brush border membrane of the midgut based on competitive and specific binding studies 61 . Interestingly, our data indicate that the resistance to Cry1Ac negatively correlates with resistance to Cry1Fa or Cry2Aa (i.e., the Cry1Ac-selected soybean loopers became more susceptible to Cry1Fa and Cry2Aa). This is one of the few empirical evidences for negative cross-resistance or negatively correlated resistance involving Bt toxins (reviewed in 29 ), deserving investigation of its genetic basis (i.e., if caused by a single or different genes 29 ), which in this case may encode altered receptor proteins interacting with these Bt toxins in their intoxication route 66 . In practical terms, negative cross-resistance may be exploited for resistance management 29 ; for example, Cry1F and Cry2A toxins may be used to preferentially kill soybean loopers that are resistant to Cry1Ac.
Although some variation exists in the patterns of cross-resistance between Cry1A and Cry2A 47,67-69 , these toxins seem to be compatible for resistance management in most pest species. Here, the clear absence of positive cross-resistance to Cry1Fa and Cry2Aa provides empirical evidence that these toxins do not share binding sites in receptor proteins 61,70 . These findings are relevant because Cry2A or Cry1F toxins are pyramided with Cry1Ac in second-generation Bt cotton 71,72 and soybean 13,14 in the Americas. Most importantly, our results indicate that Cry2A and/or Cry1F are compatible with Cry1Ac in a multi-toxin approach for resistance management of soybean looper. This is critical in the stewardship and optimal management strategy 24 of new Bt soybean varieties 13,14 that are to be introduced in the market for controlling soybean looper and other lepidopteran pests.

Material and Methods
Insect collection and rearing. Field populations of soybean looper (ca. 500 third to fifth instar larvae) were collected from non-Bt soybean (G. max) and dry beans (Phaseolus vulgaris) on farms of the Federal University of Viçosa, in Viçosa and Coimbra counties, Minas Gerais state, Brazil, in April 2014. The larvae were brought to the laboratory and reared individually on leaves of the respective host crops (i.e., non-Bt soybean or dry beans). Two to three batches of pupae (80♂ + 80♀) were placed in two polyvinylchloride cages, 20 cm diameter × 30 cm height, lined internally with sulfite paper as substrate for oviposition. Adults were fed a 10% honey solution in distilled water, and eggs were collected daily and stored in an incubator until hatching. Neonates were reared at 27 ± 1 °C, 70 ± 10% r.h. with 16:8 (light:dark) cycle on artificial diet 73 . In the F 1 generation, the larvae were divided in three subpopulations; one was selected with purified Cry1Ac toxin (1Ac-Sel), another was selected with Cry1Ac cotton leaf tissue (BG1-Sel), and a third subpopulation was left unselected (Bt-Unsel) and maintained on artificial diet using methods described above, keeping the population size at approximately 200 adults per generation. To obtain appropriate leaves for the bioassays, cotton and soybean plants were cultivated in the greenhouse following standard cultivation practices 74,75 . Cotton was sown every three months in 15-L pots with substrate composed of 3 parts of soil, 2 parts cattle manure, and 2 parts of sand to obtain plants with normal levels of Bt protein expression 52,53,76 . The plants were irrigated twice or three times a day depending on soil moisture conditions, and leaves were collected from cotton plants 45-50 d after emergence. Soybean plants were field-grown using cultivation practices as recommended for the crop, and leaves were excised when plants were in the R2-R4 growth stages 74 . Soil fertilization was as recommended for cotton 75 or soybean crops 74 . The plants were inspected weekly for mechanical pest control or disposal of infested plants when needed. Immunodetection assays using ImmunoStrip STX 74500 kit (Agdia Inc., Elkhart, IN) were used according to the manufacturer's instructions to confirm the presence or absence of the Cry1Ac trait in the Bt or non-Bt isoline plants from which foliage was excised.

Source of cotton for selection and soybean for leaf-bioassays. Transgenic plants producing Cry1Ac
Bt toxins and bioassays. The Cry1A toxins (Cry1Ab, Cry1Ac and Cry1Fa) and Cry2Aa protoxin used in the experiments were obtained from Dr. Marianne P. Carey (Case Western Reserve University, OH). The proteins were purified on HPLC, shipped as lyophilized powder, and stored at −80 °C until use, when fresh dilutions were prepared as follows. Cry1 toxins were solubilized in 100 mM Na 2 CO 3 buffer (pH 10.3, containing 10 mM DTT) and Cry2A protoxin in 50 mM Na 2 CO 3 buffer (pH 12.1, containing 5 mM EDTA and 10 mM EGTA) to produce the stock concentration (1 mg/ml) for each Bt toxin. These were further diluted with 0.1% Triton-X 100 to obtain the appropriate concentrations used in the bioassays.
All bioassays used in the selection experiment and cross-resistance study were repeated twice and included at least seven different concentrations of purified Cry toxin plus a control (i.e., 0.1% Triton-X 100 only) applied to the diet surface 77 . A single neonate (<24 h after hatching) was placed in each well of a 128 well-tray (CD International, Pitman, NJ), and held for seven days at 27 ± 1 °C, 24 h scotophase, and 80% r.h. until assessment of larval mortality 45,77 . Larvae of the Cry1Ac-selected population were not bioassayed against purified Cry1Fa as they were low in availability when the assays were conducted.
Selection experiment using purified Cry1Ac toxin. This experiment was conducted in parallel with that using Bt cotton leaf sections. Methods were adapted from Gould et al. 47 and Pereira et al. 45 . Initial bioassays to determine the population susceptibility to Cry1Ac were conducted as previously described, using the same toxin source that was later used in the selection experiment. Bioassays were done using graded Cry1Ac concentrations applied on the surface of artificial diet 77 . Larval mortality was recorded after seven days of exposure and analyzed by probit regression to determine the concentration that kills 90% of the larvae (LC 90 ), which was used as the concentration applied on the diet surface for the next generation of selection. The following Cry1Ac concentrations were used from the first to sixth generation of selection, respectively: 215, 535, 1568, 1868, 3004, and 4058 ng/cm 2 (Table S1). At least 2,500 neonates were exposed to Cry1Ac every generation of selection, and after seven days of feeding on the toxin-treated diet, the larvae with size (i.e., weight) similar to those of the control were selected. These larvae were transferred to the untreated artificial diet and reared until pupation. The adults were held in mating cages as previously described. A portion of the offspring from the parents selected in the previous generation was bioassayed as above to estimate the gain by selection. This process was repeated during six generations of selection.
Selection experiment using Cry1Ac cotton foliage. A chronic selection experiment using Cry1Ac cotton leaves throughout larval development was started in the first generation of the field-collected laboratory colony in 2014. In each generation of selection, we transferred 160 batches of 10 neonates to 16-well-plastic trays (Advento do Brasil, Diadema, São Paulo) (10 larvae/well), each well containing a cotton leaf section. After three days, we assessed the number of survivors, and they were transferred to new 16-well trays (3 larvae/well). From then on, cotton leaves were replaced every three days until pupation, when the insects were transferred to mating cages and held as previously described. The selection experiment took place during 11 generations. Soybean looper larvae were also reared in parallel on non-Bt cotton leaves to estimate natural mortality in the absence of selection. In each generation of selection, the gain by selection (i.e., increase in resistance) was estimated using bioassays with purified Cry1Ac protein as described above.
Leaf tissue assay using Cry1Ac soybean. We tested the hypothesis that the Cry1Ac resistance developed in the laboratory-selected soybean looper populations would be high enough to allow for survival on leaf tissues of the Cry1Ac Bt soybean. In a completely randomized experiment with 20 replications, we exposed larvae of the two selected and the control populations to foliage of Bt soybean (Intacta) and its non-Bt near isoline (MSOY8866, Monsanto do Brasil, São Paulo, SP). Soybean foliage was excised in the R2-R4 growth stages, quickly placed in buckets with water, brought to the laboratory, thoroughly rinsed with distilled water, and placed on paper towels to dry for 15 min at room temperature. The Cry1Ac or control soybean foliage was cut into 3-cm 2 pieces and placed in 50-ml plastic cups. Batches of 10 neonates (<1 day old) were transferred to each cup and maintained as described previously. After three days, we recorded the number of survivors and calculated the survival rate for each experimental unit (i.e., each cup, total n = 120). The total number of larvae tested in the bioassay was 1200 (400 per insect population).
Realized heritability. Following Falconer and Mackay 78 and Tabashnik 50 , we calculated the realized heritability (h 2 ) as h 2 = Response to selection ÷ Selection differential. In this equation, the response to selection (R) was calculated as: R = [Log (final LC 50 )−Log (initial LC 50 )]/n, where the final LC 50 is the LC 50 of the population after six generation of selection with purified toxin or Bt cotton foliage, respectively; the initial LC 50 is the LC 50 of the base parental population before selection, and n is the number of generations selected with Bt cotton or the purified toxin. The selection differential was calculated as follows: Selection differential = i × σ p , where i is intensity of selection calculated according to Falconer and Mackay 78 , and σ p is the phenotypic standard deviation, which was calculated as follows: σ p = [0.5 × (initial slope + final slope)] −1 . Finally, the number of generations required for a 10-fold increase in LC 50 (G) was calculated as: G = 1/R.

Statistical analyses.
The probit model was fit to bioassay data using Polo-Plus software 79 to estimate the concentration causing 50% mortality (LC 50 ) and their 95% fiducial limits as well as the slope of the concentration-response lines and their standard errors. The data were adjusted for natural mortality relative to controls when needed. Lethal concentration ratios (i.e., resistance or cross-resistance ratios) and their respective 95% confidence limits 79 were determined using the unselected control population as reference for comparison, and considered significantly different (P < 0.05) when they did not include the value one. The significance of cross-resistance between toxins was also assessed by the failure of 95% fiducial limits at LC 50 estimates to overlap, which is quite conservative 79,80 . The data from leaf tissue assays were analyzed using a two-way analysis of variance; the two main effects were insect population and plant phenotype (Bt or non-Bt soybean), both considered fixed factors. Means were separated using a comparison wise error rate (α) of 0.05 81 .

Availability of Materials and Data
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.