BPPD agrees with the following other modeling assumptions:   There are no fitness costs associated with the resistant forms of any of the three receptors. As Monsanto notes, there are recent studies in which Cry1Ac resistance is associated with fitness costs ( e. g. Bird & Akhurst 2004; Carrière et al. 2001a, b).   Resistance to each of the three Cry proteins was assumed to be complete.   The CBW model was run for each of the four regions shown in Figure 2. This provides a snapshot in time in each region.   The proportion of Bt cotton was varied for each of the three Bt cotton products across the four regions by " current" and " natural" refuge.   For CBW there were six generations of the pest per year.   The number of years until the frequency of each resistance allele exceeded 0.5 was recorded, up to a maximum of 30 years. The number of years to resistance and the relative efficacy remaining should only be interpreted on a qualitative basis.   The model was run in a simple, deterministic manner, one region at a time. Spatiallyexplicit stochastic models would, however, offer a more nuanced and dynamic picture of the interactions of the three Bt cotton products in the landscape over time.

BPPD has some concerns with the following assumptions:  42   Assumption that the initial resistance allele frequency for each of the three receptors is 0.002. Given that resistance management is more effective when the initial frequency of resistance is lower, an assumption of 0.002 should be a conservative assumption. Given the length of prior use of Bollgard cotton, 10 years, this estimation may or may not be optimistic. Monsanto's modeling does not account for any prior selection to Cry1Ac. Monitoring efforts have not detected any significant change in susceptibilities to the Bt proteins ( BPPD 2005; Tabashnik et al. 2003). Given the limitations of the sampling, probability of detection based on the sensitivity of the discriminating dose ( LC99) bioassay ( 0.01), the current resistance allele frequency for Cry1Ac is estimated to be lower than 0.01, based onn the limit of detection of the discriminating dose ( LC99) bioassay ( EPA 2001). The probability of detection of field resistance, therefore, would be relatively low unless resistance allele frequencies were high and the resistance was dominant. Given minimal adoption of WideStrike and Bollgard II and therefore minimal selection additional selection pressure for resistance to Cry1Ac, Cry1F, or Cry2Ab, it is likely that the initial resistance allele frequency of 0.002 is still a conservative assumption. Because the modeling simulations assume that all three Bt products are introduced as a mosaic, then an initial resistance allele frequency of 0.002 for Cry1Ac would be a conservative assumption. However, as noted above, given the adoption of Bollgard cotton and the selection pressure for CBW resistance to Cry1Ac, it is more likely that the resistance allele frequencies have increased and that a more relevant set of modeling scenarios would have examined prior selection ( see discussion below).   Assumption that resistance is recessive. In North Carolina, Burd et al. ( 2001) provide results from a reciprocal cross study that suggests CBW resistance to Cry1Ac and Cry2Ab is dominantly or incompletely inherited. These results suggest that the assumption that CBW resistance is recessive is perhaps incorrect. Burd et al. ( 2001) isolated nonrecessive Bt resistance genes present in CBW field populations in North Carolina and estimated the Cry1Ac resistance allele frequency for CBW as 0.0043 and the Cry2Aa resistance allele frequency for CBW as 0.00039.   Assumption that there is no prior selection for resistance to Cry1Ac. Monsanto has presumed no prior selection for CBW resistance to Cry1Ac even though Bollgard has been commercially available for 10 years. Monsanto has modeled a mosaic of all three Bt cotton products rather than a sequential introduction of Bollgard II and WideStrike following introduction of Bollgard. Zhao et al. ( 2005) have shown that the concurrent use of one and two gene Bt broccoli plants resulted in control failure of both types of Bt plants for control of Plutella xylostella ( diamondback moth) rather than the use of the two gene plants by themselves. In addition, these researchers illustrated through their modeling efforts that resistance will evolve first to the toxin that is being used singly.

Given the assumptions of the three gene model and its limitations, there is likely enough effective natural refuge to be sufficient to delay the evolution of resistance to Bollgard II cotton for more than 25 years ( not a precise number of years) under all plausible scenarios in all four regions ( Table 14). Based both on the intrinsic durability of each of the three Bt cotton products ( Figure 4) and the three gene modeling analyses for all three Bt cotton products together in the marketplace ( Table 14), Bollgard II retained the highest level of efficacy against CBW in all  43 scenarios ( all regions). This is because of the relatively high mortality of individuals heterozygous to Cry1Ac resistance in the presence of Cry2A2, as compared to WideStrike; intermediate in many scenarios because of the shared binding receptor between Cry1F and CryAc and likelihood of cross resistance, and Bollgard, weakest in all scenarios, no high dose for CBW and a single gene product. Monsanto's models predict that CBW resistance to Bollgard cotton will evolve in less than the 30 year horizon in the Georgia, Mississippi, and E. Texas regions in most scenarios except for 2 C ( Bollgard = 0.1; Bollgard II = 0.8; WideStrike = 0.1). Resistance always took at least 30 years to evolve for all three Bt cotton products in the North Carolina region in all scenarios, even the natural refuge scenarios. Monsanto's modeling does not presume any prior selection for Cry1Ac resistance.

When Bollgard cotton acreage is minimized, Bollgard II and WideStrike longevity is maximized ( Table 14). Selection pressure for resistance to Cry1Ac is reduced and the relative value of the unique proteins, Cry1F and Cry2Ab2 is enhanced. Large amounts of Bollgard II cotton in the marketplace increased the durability of both Bollgard and WideStrike ( Table 14).

TBW In Gustafson and Head ( 2005), Monsanto addressed BPPD's ( 2004b) and the SAP's ( 2004) previous concerns regarding the calculation of the effective refuge size ( weighted per host per generation), inclusion of alternate hosts ( three sub compartments for TBW vs. twocompartments increased spatiality ( four regions), impact of multiple Bt cotton products ( three gene model and scenarios varying the level of adoption of each product) and larval mortality. This means that Monsanto's current TBW modeling analysis ( Gustafson & Head 2005) is more robust than the one previously reviewed by the Agency ( BPPD 2004b) and the SAP ( 2004). As noted above, the modeling analysis provides a regional snapshot of the population dynamics of TBW and thus is a limited picture of what might be happening in the landscape ( although these snapshots may represent the worst case scenario). Making an extrapolation over many years based on just a few snapshots in time, given the variability of the data, is uncertain.

Monsanto combined pooled, county level estimates of the percent cotton reared TBW moths with county level landcover information to estimate the current effective refuge and the natural refuge for each county by month ( see Head & Gustafson 2005, discussed in Section II and III above). The landscape is divided into three sub compartments to estimate TBW current and natural effective refuge: Bt cotton, non Bt cotton, non cotton area ( Figure 1). The current effective refuge for TBW is defined as the proportion of TBW moths actually produced in the effective refuge compartment prior to selection by Bt cotton ( Equation 9). The potential natural refuge for TBW is given by Equation 10; it is conservatively assumed that all current non Bt cotton acres would contain cry1Ac so the term, ANBTC , is removed. Table 9 contains the current and natural effective refuge values used in the TBW modeling. These were the averages of all available 2004 and 2005 values for the selected counties where the number of moths collected during that month were at least 10. BPPD agrees with Monsanto's method for calculating the relative TBW productivity on each prospective alternate host. However, uncertainties in the pheromone sampling, gossypol analyses, and spatial landscape mapping affect the estimation of  44 the current and natural effective refuge. These issues were discussed in Sections II and III of the review.

BPPD agrees with the following assumptions Monsanto made for the three gene model based on the mechanisms of activity of Bollgard, Bollgard II, and WideStrike cotton ( i. e. expected receptor binding patterns of Cry1Ac, Cry1F, and Cry2Ab2 to TBW midgut membranes). It is assumed that the primary mechanism of Bt resistance is associated with modifications to the receptor binding site.

  The Cry1Ac toxin, present in all three products, binds to two receptors, 60% to receptor A and 40% to receptor B.   The Cry1F toxin, present only in WideStrike cotton, binds exclusively to receptor A.   The Cry2Ab2 toxin, present only in Bollgard II cotton, binds exclusively to receptor C.

As noted earlier, these general assumptions were evaluated by the June 2004 SAP for a spatiallyexplicit stochastic CBW resistance model used by DAS for its WideStrike cotton product and found to be acceptable ( SAP 2004). See discussion above regarding Bt resistance mechanisms.

BPPD has some concern about whether these assumptions are still valid for TBW. BPPD reviewed the relevant literature and determined that three site model proposed by Jurat Fuentes & Adang ( 2001) for binding of Bt Cry1 toxins to sites in the TBW midgut membrane is relevant to this discussion. Based on the ligand blot analyses, the Cry1Ac toxin was able to recognize three receptors: A, B, and C; while the Cry1Fa toxin was able to recognize receptor A and perhaps a second predicted, but not determined site. Receptor C is only recognized by the Cry1Ac toxin. Neither Cry1Ea nor Cry2a competed with any of the labeled Cry1A toxins. Cry1Ac competed with high affinity for Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for Cry1Ab binding sites. Cry1Ab competed for labeled Cry1Ac binding sites with a higher affinity than did Cry1Aa, Cry1Fa, and Cry1Ja. It was noted by the study authors that it was inherently difficult to label Cry1Fa. One can conclude from Fuentes and Adang ( 2001) that Cry1F and Cry1Ac have shared binding affinity for Receptor B ( in their model) which is analogous to what was proposed by Monsanto in its three gene model ( competition is for receptor Ai in this model) ( Gustafson & Head 2005). Some level of cross resistance between Cry1Ac and Cry1F associated with a common resistance mechanism of reduction in receptor binding would be expected. This issue is of particular concern for WideStrike cotton because it expresses both Cry1F and Cry1Ac and there is competition between Cry1F and Cry1Ac for a shared receptor. Competition binding experiments indicate that perhaps 40% of the labeled Cry1Ac binding to brush border membrane vesicles was reduced by competition with Cry1Fa, although a saturation concentration of Cry1Fa was not reached. Based on the data provided in Jurat Fuentes & Adang ( 2001), Monsanto's assumptions regarding the binding patterns of Cry1Ac, Cry1F, and Cry2Ab2 to receptors A, B, and C on TBW midgut membranes are acceptable with one caveat. Additional competition binding studies using TBW brush border membrane vesicles at saturable concentrations of Cry1Fa are recommended. Based on the results of additional competition binding studies, the survival probabilities of all 27 genotypes may need to be recalculated and the modeling redone. Uncertainty in the shared binding affinity  45 of the Cry1Ac protein to receptor A and B ( in Monsanto's model) would impact the modeling predictions for all three Bt cotton products because the Cry1Ac protein is common to all of them. Monsanto's modeling does not assume any prior selection for Cry1Ac resistance. The degree to which the modeling predictions would be impacted is unknown The June 2004 SAP raised an issue as to how DAS calculated the expected mortality of different genotypes as a result of exposure to Cry1Ac, the factor " Z" ( SAP 2004). Monsanto has modified the mathematical expression of the competition between Cry1F and Cry1Ac for receptor A and the calculation of the expected mortality of different genotypes as a result of exposure to Cry1Ac. The survival probabilities of all 27 TBW genotypes are found in Tables 11. To model for resistance, Monsanto assumed that each receptor has an alternate, fully resistant form whose structure is determined by a single, diallelic expression in a heterozygote is additive such that a heterozygote has 50% susceptible and 50% resistant receptors present in the gut. The concentration of the toxin receptor complex is such a heterozygote is then half ( given by Equation 12) and then this concentration is entered in Equation 11 to determine the quantitative impact on the probability of survival due to that particular toxin receptor combination. These values differ from those reported by DAS for the same three products ( SAP 2004) because of differences in the mathematical calculations.

BPPD agrees with the following other modeling assumptions:   There are no fitness costs associated with the resistant forms of any of the three receptors. As Monsanto notes, there are recent studies in which Cry1Ac resistance is associated with fitness costs ( e. g. Bird & Akhurst 2004; Carrière et al., 2001a, b).   Resistance to each of the three Cry proteins was assumed to be complete.   The proportion of Bt cotton was varied for each of the three Bt cotton products across the four regions by " current" and " natural" refuge.   For TBW, the model was run for the " worst case" county in each state ( indicated by shading in Tables 7 and 8).   For TBW were six generations of the pest per year.   The number of years until the frequency of each resistance allele exceeded 0.5 was recorded, up to a maximum of 30 years. The number of years to resistance and the relative efficacy remaining should only be interpreted on a qualitative basis.   The model was run in a simple, deterministic manner, one region at a time. Spatiallyexplicit stochastic models, however, would offer a more nuanced and dynamic picture of the interactions of the three Bt cotton products in the landscape over time.

BPPD has additional concerns regarding the assumptions for: initial resistance allele frequency for each of the three receptors and prior selection for Cry1Ac resistance. These were discussed above in the context of CBW modeling but, in general, are applicable also to the TBW modeling with the addition of the following comments.   Assumption that the initial resistance allele frequency for each of the three receptors is 0.002. Gould et al. ( 1997) estimated the TBW resistance allele frequency as 0.001. Given the length of prior use of Bollgard cotton, 10 years, this estimation may or may not  46 be optimistic. Monitoring efforts have not detected any significant change in susceptibilities to the Bt proteins ( BPPD 2005; Tabashnik et al. 2003). As noted above, given the limitations of sampling and sensitivity of detection ( 0.01), the probability of detection of field resistance, therefore, would be relatively low unless resistance allele frequencies were high and the resistance was dominant.   Assumption that resistance is recessive. Because Cry1Ac, Cry2Ab2, and Cry1F are all expressed at a " high dose" in all three Bt cotton products, it is expected that resistance will be conferred by rare, recessive alleles and most resistant adults from Bt crops will mate with susceptible adults from refuges. In a recent review, Tabashnik et al. ( 2003) comment that "  inheritance of resistance to transgenic plants with high concentrations of Bt toxins is recessive in all cases examined to date involving pests that have high inherent susceptibility to the toxins produced ".   Assumption of no prior selection to Cry1Ac. See earlier BPPD comments on CBW modeling.

From a qualitative perspective, modeling indicates the intrinsic durability of Bollgard II is greater than WideStrike which is greater than Bollgard. The intrinsic durability of all three Bt cotton products is much greater for TBW than for CBW because of the " high dose" of Cry1Ac for TBW expressed in all three products. In virtually all cases, all three products retained their efficacy ( i. e. no resistance) for more than 30 years ( maximum time for the simulation) even if all cotton in a region is planted to that product and no structured refuge is required ( i. e. all natural refuge) ( see Figure 5). The only exceptions occur for Bollgard cotton in Tennessee and Mississippi. Given the assumptions of the three gene model and its limitations, there is likely enough effective natural refuge to be sufficient to delay the evolution of resistance to Bollgard II cotton for more than 30 years ( i. e. the time horizon of the model, not to be interpreted as a precise number of years) under all plausible scenarios in all four regions ( see Table 15). This is due to the extremely high efficacy of Cry1Ac against TBW, and the fact that Cry1Ac is present in all three Bt cotton products. In the state with the lowest natural refuge for TBW, Mississippi ( see Table 13), resistance to Cry1Ac and Cry1F evolved after 21 years in scenario 1 N if the structured refuge requirements for Bollgard and WideStrike cotton were removed. Uncertainties in the pheromone captures, gossypol analyses, spatial analysis, effective refuge calculation, degree of shared binding affinity of Cry1Ac to receptor A and B, effect of prior selection for Cry1Ac resistance, and other modeling assumptions affect the precision and accuracy of the modeling predictions.

Caprio ( 2006) used a spatially explicit, stochastic, two locus resistance model to investigate the likelihood that TBW would not evolve resistance to Bollgard II within a 15 year time horizon. He used two different scenarios, one with a 4% structured refuge and 1% wild hosts ( 5% untreated refuge), and the second with no structured refuge and a 1% wild host refuge. The model simulated 1024 cotton patches, each ca. 100 acres in size. The model assumed that only Bollgard cotton was used initially and simulated a gradual transition to Bollgard II over five years. Mortality rates for this cotton with nine different genotypes were calculated. The initial gene frequency was assumed to be 0.001. The model assumes four generations of TBW develop on cotton per year. Generations outside of cotton were not incorporated into the model. A  47 carrying capacity of 80,000 larvae ( eggs/ patch) for early pre bloom cotton, 800,000 for peak bloom cotton, and 500,000 larvae ( eggs/ field) for flowering cotton were used in the modeling. Fitness costs were not included in the model. The model parameters were not varied. Thirty runs of the simulation for each of the two scenarios were made and changes in resistance allele frequencies at both resistance loci were evaluated. In 30 runs of the simulation with a structured 5% untreated refuge, resistance was not observed at either locus. The mean rate of increase in resistance allele frequency for the Cry2A2 locus was 1.032 ( SD = 0.151), while the mean rate of increase for the Cry1Ac locus was 1.0567 ( SD= 0.169). In 30 runs of the simulation with no structured refuge and a 1% unstructured refuge, resistance was observed to occur in three cases to the Cry1Ac trait in Bollgard II. The mean rate of increase in resistance allele frequency for the Cry2Ab2 locus was 0.8984 ( SD = 0.391), while the mean rate of increase for the Cry1Ac locus was 85.88 ( SD= 262.53; three outlier populations). These results suggest that the risk of TBW resistance in a 15 year time horizon with or without a structured refuge is relatively small if the estimates of mortality of TBW larvae and other components of the model are reasonably accurate. The results do suggest much higher risk of TBW resistance to the Cry1Ac trait if the structured refuge is removed. The predictions of Caprio's spatially explicit, stochastic, twolocus TBW resistance model are similar to those predicted by Monsanto's three gene, deterministic model ( Gustafson and Head, 2005). That is, structured and natural refuge delay TBW resistance effectively to the two proteins, Cry1Ac and Cry2Ab2, expressed in Bollgard II.

Overall Conclusions and Discussion Overall, the modeling suggests that Bollgard II cotton should have more than 25 years of durability for the control of CBW and TBW in all regions with natural refuge as the only source of susceptible insects. The presence of larger amounts of Bollgard II in the marketplace also increases the durability of other Bt cotton products that are present but the intrinsic durability of Bollgard II cotton is much greater than Bollgard or WideStrike cotton. However, there are uncertainties in the modeling parameters and assumptions which could impact the modeling output.

Modeling suggests that the overall durability of Bollgard II cotton can be enhanced if Bollgard cotton is removed from the marketplace. This conclusion is supported by other researchers who examined the benefit of managing resistance evolution to two toxins with dissimilar modes of action using a pyramided approach ( Zhao et al. 2005; Roush 1998; Livingston et al. 2004; Hurley 2000; Caprio 2006). On the other hand, the concurrent use of single and two gene Bt plants can offer exposed populations a " stepping stone" to develop resistance to both proteins. That is why it is important to consider the removal of single, less durable, Bt cotton products from the marketplace in favor of more durable, two gene ( assuming independent modes of action) products. In Australia, the use of the single gene Bt cotton product ( i. e., Bollgard) and the pyramided Bt cotton product ( i. e. Bollgard II) was permitted only for the first two years after the introduction of Bollgard II, but now only Bollgard II is permitted. In the U. S., Bollgard II cotton has been in the marketplace for four years ( registered in December, 2002) and WideStrike cotton has been in the marketplace for two years ( registered in September, 2004). In 2004, Bollgard cotton acreage accounted for > 95% of all Bt cotton acreage in the U. S. ( see Head et al.  48 2005, MRID# 467172 03). Ten years of selection pressure for resistance to Cry1Ac has already occurred. Field resistance to Cry1Ac places additional selection pressure on the Cry2Ab2 component of Bollgard II cotton. Monsanto's modeling does not assume any prior selection for Cry1Ac resistance either by TBW or CBW. Encouraging the adoption of Bollgard II will increase the overall durability of all three Bt cotton products. From an insect management point of view, removal of Bollgard cotton from the marketplace should be encouraged as quickly as possible.

Researchers from Australia ( Mahon et al. 2004) have reported that one population of Helicoverpa armigera ( a close relative of Helicovera zea, CBW, in the U. S.) collected from maize plants exhibited resistance to Cry2Ab. These researchers note that there is no evidence of cross resistance to Cry1Ac and that inheritance of resistance is recessive. An explanation as to why H. armigera had unexpected high levels of Cry2Ab2 resistance levels is unknown, but research is ongoing to evaluate this resistance. A better understanding of background levels of resistance to Cry2Ab toxins in H. armigera populations and opportunities for survival of resistant genotypes on Bollgard II is needed. What this means for either TBW or CBW resistance to Cry2Ab toxins in Bollgard II in the U. S. is not known, but we should be aware of the possibility that Cry2Ab resistance may be more common than expected.

V. Proposed Revisions to the Bollgard II IRM Program Monsanto's proposed revisions to the IRM program to use natural refuge for Bollgard II cotton are contained in the volume titled " Scientific and Economic Justification for Not Requiring Structured Cotton Refuges for Bollgard II Cotton in the U. S. Cotton Belt from Texas to the East Coast" ( MRID# 467172 03). This report summarizes the alternate TBW host studies, TBW effective refuge, and resistance modeling previously discussed in this review. In addition, Monsanto has included sections describing cross resistance potential, CBW considerations, economic and environmental benefits of the proposed IRM revision.

a) Natural Refuge Proposal As described in the Background section, the terms and conditions of registration for Bt cotton varieties ( including Bollgard and Bollgard II cotton) required the planting of a structured, non Bt cotton refuge. For TBW, the refuge strategy is supported by " high dose" expression of Bt toxin( s) by the registered hybrids. For CBW, although there is not high dose expression, the pest is highly polyphagous and experimental data have shown that it makes use of alternate, noncotton hosts ( see discussion in the Background section). The IRM plan for Bt cotton also includes resistance monitoring requirements for both TBW and CBW on all registered toxins. As of the 2004 growing season, no documented cases of resistance of significant shifts in pest susceptibility to Cry1Ac ( the toxin in Bollgard) have been detected ( see BPPD 2005).

Monsanto has proposed to replace the structured refuge requirements for Bollgard II cotton with a natural refuge approach ( a similar proposal for Bollgard cotton has not been proposed).  49 Growers would no longer be required to plant a non Bt cotton refuge and could plant up to 100% Bollgard II cotton. The natural refuge strategy would be applicable to the southeastern cotton growing regions including Texas ( western cotton regions, in which pink bollworm is the primary target pest, are not included). This proposal is based on a number of supporting factors: 1) Bollgard II expresses two distinct Bt toxins which offers an IRM advantage over single toxin products; 2) Bollgard II offers better control of the target pests ( TBW and CBW) than Bollgard cotton; 3) The alternate host data for TBW ( reviewed in this memorandum) and CBW ( previously reviewed  see Background section) indicate that adequate natural refuge exists independent of non Bt cotton refuges.

Monsanto's submission described the potential resistance management benefits of using multiple toxins ( also known as " pyramiding" toxins). A number of models ( Roush 1994, 1998 and Caprio 1998b) indicate that the deployment of multiple toxins in a transgenic crop can delay the onset of pest resistance longer than a single toxin crop. The advantages of pyramided toxins are contingent on several factors: high efficacy of each toxin against the target pest and distinct modes of action for each toxin ( i. e. lack of cross resistance). In the case of Bollgard II, the Cry1Ac and Cry2Ab2 Bt toxins are expressed ( the event was created by inserting the Cry2Ab2 gene into existing Bollgard cotton). Both Cry1Ac and Cry2Ab2 each are known to have high activity against TBW and together the toxins were shown to increase the overall efficacy against TBW by 3.5 fold. For CBW, Cry2Ab2 was shown to have high activity ( close to " high dose"), while Cry1Ac has less activity and is not considered high dose.

b) Cross Resistance The IRM benefits of a multiple gene product could be reduced if there is cross resistance between the expressed toxins. Cross resistance can result if a pest develops resistance to one toxin, which then confers a degree of resistance to the second toxin. Monsanto's submission summarizes information to support the contention that there is no cross resistance potential between Cry1Ac and Cry2Ab2. These summarized data to discuss cross resistance were originally submitted by Monsanto with the initial registration application for Bollgard II ( MRID# 455457 01).

Structurally, the Cry1A and Cry2A groups of Bt toxins are divergent, with only 20% similarity between amino acid sequences. In addition, polyclonal antibody assays showing an absence of cross reactivity of anti Cry2Ab2 antibodies with Cry1Ac protein ( and vice versa) demonstrate that the tertiary structure of Cry2A proteins differs significantly from Cry1A proteins. Given the lack of structural similarities, Monsanto concluded that the insecticidal mechanisms of Cry1Ac and Cry2Ab2 are distinct and unlikely to result in cross resistance.

Once ingested by the target pests, the Cry1Ac and Cry2Ab2 toxins behave differently. During trypsin digestion, Cry1Ac forms a stable core protein that is not produced by Cry2Ab2 under similar conditions. Also, in the insect midgut, the two toxins bind to the gut membranes in different manners: Cry1Ac interacts with brush border membrane proteins to open ion channels whereas Cry2Ab2 interact with other proteins to open unique ion channels. The ion channels  50 created by Cry2Ab2 are larger and have reduced ion selectivity than those created Cry1Ac, which may explain the increased potency observed with the toxin. As with the structural differences between the two toxins, these differences in membrane interaction suggest low cross resistance potential between for the Bollgard II toxins.

Further evidence to document the lack of cross resistance potential was shown in studies utilizing Cry1Ac resistant colonies of TBW and CBW. When challenged on Cry2Ab2 ( Bollgard II plant material), Cry1Ac resistant TBW remained susceptible to the protein with few survivors ( none of the survivors progressed beyond the first larval instar). A similar experiment with CBW resulted in less than 5% survival on Bollgard II cotton plants.

BPPD Review ( Cross Resistance)

Cross resistance data were previously submitted to BPPD by Monsanto for the initial registration of Bollgard II. These data ( the same data set described above) were reviewed in detail by BPPD ( see BPPD 2002a) prior to the issuance of the registration. In this review, BPPD agreed with Monsanto's overall conclusions that the cross resistance potential between Cry1Ac and Cry2Ab2 in Bollgard II should be low for both TBW and CBW based on structural and binding characteristics.

However, since Bollgard II has been registered, additional research has been conducted into the cross resistance potential of Cry1 and Cry2 proteins. In one study, Jurat Fuentes et al. ( 2003) investigated resistance to Cry1Ac and Cry2Aa in TBW. Several strains of TBW that had been selected on and developed resistance to Cry1Ac were found to also be resistant to Cry2Aa. Because these two toxins are not known to share binding sites in TBW, the authors suggested that another mechanism may be involved in the observed resistance. This alternate resistance mechanism could be one that affects a shared process in the mode of action of the two toxins. As was described by Monsanto, the structure and binding properties of Cry1A and Cry2A toxins are different at the membrane level. However, both toxins have similar activation processes in the midgut before membrane interaction; an alteration of the toxin activation process ( i. e. differential midgut protease activity) could lead to common resistance to both proteins. The authors also suggested " midgut epithelium regeneration" as a potential mechanism ( in which the midgut recovers from exposure to cry toxins), although this area requires additional research.

Though Cry2Aa is not a toxin expressed in Bollgard II, the work of Jurat Fuentes et al. ( 2003) illustrate the need to consider alternate resistance mechanisms other than structure or binding when evaluating cross resistance potential. Other potential resistance mechanisms could include metabolic changes ( e. g. protease inhibition, gut recovery) or behavioral adaptations. Such alternate mechanisms may be complex and difficult to evaluate in the context of transgenic crops ( most cross resistance evaluations have been conducted in the laboratory). BPPD previously discussed some of the theoretical considerations for cross resistance in transgenic crops as part of the 2001 Bt crops reassessment ( see EPA 2001), concluding that additional study is needed to full understand the ramifications for IRM.  51 For Bollgard II, the data evaluated to date on protein structure and midgut binging seem to indicate that the cross resistance potential may be relatively low for Cry1Ac and Cry2Ab2. However, based on the work of Jurat Fuentes et al. and the limited overall knowledge base on the subject, cross resistance cannot be entirely eliminated as a possibility with Bollgard II. Another important consideration is the presence of Bollgard cotton ( or other single gene varieties expressing Cry1Ac) in the marketplace with Bollgard II. If resistance to Cry1Ac were to develop on Bollgard, dual gene Bollgard II would essentially become a single toxin ( Cry2Ab2) product. In this case, TBW would only require a single resistance mechanism to Cry2Ab2, negating the benefits of Bollgard II as a pyramided crop. Monsanto's natural refuge proposal made no specific commitment to withdraw Bollgard from the market should the proposal be accepted, so it is likely significant acreage of Bollgard will remain in place.

c) Alternative Hosts/ Natural Refuge  CBW CBW is commonly known as a highly polyphagous pest that feed on a wide range of plant hosts, including cultivated crops, wild host plants, and weeds in addition to cotton. To evaluate and quantify these alternate hosts as potential natural refuge, Monsanto conducted a large scale experiment during 2002 and 2003. This study involved an aerial mapping project of cropping patterns in cotton growing states, a survey of CBW production on alternate hosts, and a bioassay designed to detect the type of host plant used for development of sampled CBW ( a " C3/ C4" analysis of carbon isotopes). The C3/ C4 bioassay is described in the Background section of this review.

Overall, Monsanto concluded that these studies showed that there are substantial areas of alternate host crops ( corn, peanut, sorghum, and soybean) available in cotton growing areas and that CBW production is high on these crops. The C3/ C4 assays revealed that C4 alternative hosts ( primarily corn and sorghum) make a significant contribution to the CBW adult population throughout the season. These data have been previously reviewed by BPPD ( see BPPD 2004a) and are fully discussed in the Background section. BPPD previous reviewed Monsanto's model as part of the June 2004 SAP meeting on the effective of natural refuge for CBW resistance management in Bollgard and Bollgard II cotton ( SAP 2004). Monsanto revised its current and natural effective refuge calculations and model based on the June 2004 SAP recommendations ( SAP 2004). Based on Monsanto's modeling of intrinsic durability and three gene multiple product durability modeling, there is likely enough effective natural refuge to be sufficient to delay the evolution of CBW resistance to Bollgard II cotton for more than 25 years ( not a precise number of years) under all plausible scenarios in all four regions. Bollgard II retained the highest level of efficacy against CBW in all scenarios ( all regions) because of the relatively high mortality of individuals heterozygous to Cry1Ac resistance in the presence of Cry2A2, as compared to WideStrike; intermediate in many scenarios because of the shared binding receptor between Cry1F and CryAc and likelihood of cross resistance, and Bollgard, weakest in all scenarios, no high dose for CBW and a single gene product. The presence of greater amounts Bollgard II cotton in the marketplace increased the durability of both Bollgard and WideStrike cotton. Conversely, the presence of substantial amounts of Bollgard cotton in the marketplace tended to decrease the longevity of Bollgard II and WideStrike cotton. Monsanto's modeling did  52 not assume prior selection for Cry1Ac resistance. A detailed discussion of the CBW modeling is found in Section IV of this review.

d) Alternative Hosts/ Natural Refuge  TBW Like CBW, TBW is known as a polyphagous pest that utilizes a number of crops and wild hosts for development. To further analyze the potential of alternate hosts to serve as refuge, Monsanto provided a literature review of TBW biology, a description of a two year TBW sampling effort/ gossypol bioassay to determine host plant development, a calculation of TBW " effective" refuge, and the results from an aerial mapping project to investigate cropping patterns. From these efforts, Monsanto has concluded that a large portion of the TBW population in cottongrowing regions originates from alternate ( non cotton) hosts, resulting in an effective refuge that exceeds 20%. Monsanto's submitted materials and conclusions are reviewed and discussed in detail in sections I, II, III, and IV of this document.

e) Modeling   TBW and CBW As part of the analysis of alternate host utilization and the impact on IRM/ refuge, Monsanto ran model simulations using the alternative host data obtained for CBW and TBW. Simulations were run for distinct cotton growing regions ( CBW) or states ( TBW) with scenarios accounting for both natural refuge only and effective refuge ( natural refuge plus Bt cotton refuges and other non Bt cotton acreage). For TBW, " worst case" counties ( those with the lowest amount of natural refuge) were modeled. For CBW, the four regions were modeled. Based on the model results, Monsanto concluded that Bollgard II cotton would have more than 25 years without CBW or TBW resistance using the natural refuge strategy. None of three Bt cotton products lost efficacy against TBW during the 30 year time horizon of the modeling using any current or natural refuge scenario except scenario 1 N, if the structured refuge requirements for Bollgard and WideStrike cotton were removed. CBW modeling predicted that higher amounts of Bollgard II enhanced the durability of both Bollgard and WideStrike. Conversely, higher amounts of Bollgard reduced the durability of both Bollgard and WideStrike. The modeling and results are discussed in detail in section IV of this review.

f) Economic Benefits Monsanto's submission concluded that replacing the structured refuge requirements for Bollgard II will result in economic benefits to growers. Overall, Bollgard II cotton provides higher yields and less input costs compared with non Bt ( conventional) cotton treated with insecticides. Under the current IRM requirements ( 5% or 20% non Bt cotton refuges), growers experience refugerelated costs including reduced productivity in refuge acres in addition to refuge maintenance requirements ( time and labor).

For the 20% refuge requirement, growers must plant the required non Bt cotton acreage within one mile of the Bollgard II acreage and may treat the refuge as needed with insecticides to control lepidopteran pests. Comparative studies summarized by Monsanto have shown that the  53 pest control costs are smaller ( under moderate to high pest pressure) and crop yields are higher with Bt cotton relative to conventional cotton. For example, data from 1995 2002 showed an average increase in yield ( lint) of 6% with Bollgard cotton ( another set of data showed a 10% average lint increase during 1995 1999). Since refuge acres are essentially conventional cotton that is managed with insecticides, growers do not experience these benefits of Bt cotton on at least 20% of their cotton acres. One study cited by Monsanto ( Banerjee and Martin 2005) demonstrated that Bt cotton had a $ 38 per acre benefit over conventional ( refuge) cotton plantings. Other data sets tabulated by Monsanto ( 1995 2002) and a third party study ( 1995 1999) indicated that growers received an average net dollar return of $ 39.35/ acre and $ 49.80/ acre respectively with Bollgard. Based on these results, Monsanto estimated that Bt cotton provides a $ 40/ acre benefit over the 20% treatable refuge acreage, not including other refuge management costs ( e. g. scouting, labor, time, etc.). Therefore, grower using this refuge option will lose $ 40 per acre on 20% of their cotton acreage; when averaged across all cotton acres ( Bt and refuge), the cost amounts to $ 8 per acre. For a typical grower planting 1,800 acres ( the average size farm in the Delta region) the 20% refuge option results in a total cost of about $ 14,400 ( 1,800 acres x $ 8 per acre cost).

With the 5% refuge options ( embedded or external unsprayed), Monsanto estimated that growers can lose up to $ 150 per acre relative to their Bollgard acreage. Another estimate from a cotton researcher estimated losses of $ 67 per acre with the 5% options ( Martin et al. 2006). The losses can be attributed primarily to reduced yields and are particularly acute for the 5% unsprayed option, where as much as 100% of the crop can be lost due to pest infestation. Monsanto calculated that growers planted an unsprayed refuge will yield almost 250 pounds per acre less than conventionally grown ( insecticide treated) cotton. For the 5% refuge, the total cost when averaged across all cotton acres ( 5% refuge and 95% Bt cotton) is $ 7.50/ acre ( similar to the $ 8/ acre cost for the 20% refuge). Considering a typical 1,800 acre cotton farm, the overall cost is approximately $ 13,500 ( 1,800 acres x $ 7.50 per acre cost).

Monsanto's submission also discussed other non quantified refuge related costs to growers ( i. e. non yield and treatment related). In much of the cotton growing area, farms are becoming larger and more complex. Cotton plantings may include many fields over large areas, with some acreage falling in multiple counties. The need to plant refuges can limit the flexibility of these operations by requiring in depth land use planning. During planting, refuges add logistical impediments, such as the use of additional labor and the need to clean planter boxes between fields.

BPPD Review ( Economic Benefits)

As part of the evaluation of the Bollgard II registration, BPPD reviewed benefits information related to the product ( BPPD 2002b), including an assessment of the economic benefits of the crop. BPPD estimated the per acre benefit of Bollgard II to be $ 27.63 per acre, considering the ( projected) technology fee, adoption rate, improved efficacy/ pest spectrum, and reduced insecticide costs for secondary pests ( e. g. beet armyworm). This figure differs from Monsanto's calculations, which were $ 40 to $ 150 per acre, depending on the refuge option employed. It is  54 noted that BPPD's assessment was conducted prior to registration using projections for many of the inputs, while Monsanto used or cited data obtained from farm surveys of growers planting Bollgard. Regardless of the specific per acre benefit, BPPD agrees that Bollgard and Bollgard II provide significant economic benefits to growers relative to conventional and refuge non Bt cotton.

On the other hand, short term economic benefits to growers may be compromised should resistance to Bt cotton occur. The primary objective of IRM and structured refuges has been to preserve the effectiveness of Bt toxins and maintain long term benefits to growers. If resistance were to occur, growers will likely experience costs related to reverting to other ( still efficacious) Bt varieties or conventional ( insecticide treated) cotton. Additional costs could result from implementing the remedial action plan, which requires ( among other measures) the use of conventional insecticides to control outbreak pest populations. Monsanto's submission did not address the potential effects of resistance on the cost/ benefit analysis for Bollgard II, though quantifying such costs would likely be difficult and involve imprecise estimates.

g) Environmental Benefits In addition to direct economic benefits to growers, Monsanto's submission described a number of environmental benefits resulting from adoption of Bollgard II cotton. These benefits include improved resistance management, reduced use of conventional insecticides, and prolonged efficacy of conventional insecticides.

In terms of IRM benefits, Bollgard II provides a two toxin approach to controlling the target pests. Models have shown that a dual dose product can delay resistance relative to single dose products, although this advantage can be curtailed if significant acreage of single toxin products also coexists with the dual toxin product in the landscape. Single gene products ( e. g. Bollgard cotton) can provide a " stepping stone" to resistance, increasing the frequency in which resistance develops in the dual gene transgenic crop. To date, Bollgard cotton has much greater adoption among cotton growers than Bollgard II, largely due to economic reasons ( i. e. Bollgard II has no clear advantage over Bollgard for most growers). However, Monsanto has contended that should the natural refuge proposal be accepted, growers will have a clear incentive to adopt Bollgard II cotton on a wider scale ( i. e. no required structured refuge, increased economic benefits). If this proves to be the case, the transition from Bollgard to Bollgard II across the landscape will improve IRM by reducing selection pressure to Cry1Ac found in single toxin Bollgard hybrids.

Further insecticide use reduction benefits could be obtained through the adoption of the natural refuge proposal ( allowing Bollgard II on 100% of growers' acreage). A survey cited by Monsanto showed that 22% of cotton growers utilized the 20% refuge option, which allows for the use of conventional insecticides to treat pests in the refuge. By planting 100% Bollgard II, growers would no longer need to treat those 20% refuge acres for lepidopteran pests. Based on the 22% adoption figure, Monsanto estimated that 385,000 total refuge acres could be saved from insecticide treatment. Monsanto also noted that Bollgard II cotton has the potential to reduce up to three insecticide treatments per season in comparison to conventional cotton. The  55 resulting decrease in pesticide use has potential benefits for non target species, including predatory arthropods that would avoid exposure to non specific insecticides. Additional benefits could result from reduced fuel use and the need to dispose of fewer pesticide containers.

In conjunction with overall reduced pesticide usage, Monsanto has proposed that the efficacy of conventional insecticides could be preserved for longer periods of time. Less use of pesticides would mean less exposure to pest populations and ultimately less selection pressure for resistance. Pest resistance has been problematic for a number of chemicals used in cotton, most notably the pyrethroid insecticides and TBW.

BPPD Review ( Environmental Benefits)

BPPD's assessment of the benefits for Bollgard II identified both resistance management and reduced insecticide use as positive environmental advantages of the product. For IRM, it is clear that a two gene product with functional high doses for the target pests is a better option than a single gene product for the same pests. However, as Monsanto has stated in their report, a landscape containing a mixture of single gene and dual gene products can compromise the IRM benefits of the two gene crop. This can occur if resistance develops to the single gene crop ( Bollgard/ Cry1Ac in this case); the dual gene product ( Bollgard II) essentially becomes a single gene product expressing Cry2Ab2. Monsanto's natural refuge proposal provides no details or plan for increasing adoption of Bollgard II ( and phasing out Bollgard) other than the incentive to forgo structured refuge with Bollgard II. It is not clear how quickly Bollgard II acreage will increase and whether sufficient hybrids will be available with the Bollgard II genes. Given the wide discrepancy between Bollgard and Bollgard II acreage, it is highly probable that significant acreage of Bollgard will remain in place even if the natural refuge proposal is implemented. Since large acreage of Bollgard could present a resistance risk for Bollgard II, it is recommended that Monsanto provide details as to how the adoption of Bollgard II/ phase out of Bollgard will be facilitated.

BPPD agrees with Monsanto regarding the benefits of reduced pesticide use. Bt cotton has clearly decreased the need for insecticide treatments for lepidopteran pests ( refer to the benefits assessment in EPA 2001). The use of 100% Bollgard II, with high efficacy against the major target pests and some secondary pests, presumably would eliminate the need for treatment of the 20% structured refuge option. The exact amount of insecticide reduction would be dependent on the scale of adoption of Bollgard II, an issue that remains unclear. It should be noted that, like the economic and IRM benefits, is pest resistance occurs to Bollgard II, the pesticide use reduction may be compromised. In fact, resistance would likely result in a substantial increase in the amount of pesticide used, as growers would either need to treat Bollgard acreage ( as part of remedial action) or resume planting conventional cotton varieties and regular pest treatments.

BPPD Review ( Overall Natural Refuge Proposal for TBW)

Taken together, the TBW sampling, gossypol analyses, and effective refuge determinations clearly demonstrate that a significant portion of the TBW population is derived from non cotton  56 hosts. This conclusion supports what is commonly known in the literature: that TBW is a polyphagous pest that utilizes a number of cultivated crops, wild hosts, and weeds for larval development. However, questions remain on the larger issue of whether these alternate hosts can serve as unstructured refuge for TBW for Bollgard II Bt cotton. Within the Cotton Belt, the gossypol data differed greatly by state/ region. For some areas ( i. e. North Carolina/ Georgia), the proportion of non cotton origin TBW was consistently high throughout the season and may support the natural refuge proposal. In other regions ( i. e. the Mississippi Delta, eastern Texas) the data were variable and more difficult to interpret. Given the assumptions of the Monsanto model, TBW resistance to Bollgard II is predicted not to evolve over the 30 year time horizon of the model for any scenario ( current or natural effective refuge) in any region. On the other hand, there are a number of unresolved issues with the data, effective refuge, modeling, and interpretation of the results ( detailed below). Should these issues be resolved, BPPD could support the use of natural refuge alone for Bollgard II in the southeastern U. S. cotton region. The strongest case for support of a natural refuge for TBW resistance management exists for the North Carolina and Georgia regions.

While Monsanto utilized the same gossypol methodology in both 2004 and 2005, there was no statistical analysis of the gossypol results between the two years, likely because of differences in sampling for each season. Therefore, it is not possible to compare the results between seasons except on a qualitative basis. Though many of the state trends were similar in both seasons, it is not known if there was a definitive correlation between the data sets.

As previously stated, the results from the gossypol bioassays varied by region. Perhaps the strongest case for the use of natural refuge can be made in North Carolina and Georgia. In these states, the data were more consistent and clearly showed that the large majority (> 90%) of TBW originated from non cotton sources. This trend was clear throughout the entire cotton growing season in both 2004 and 2005 ( see table 3 in this review or Monsanto figure 1 in appendix 1). These states are known to contain substantial acreage of alternate crops ( peanut, tobacco, and soybean) that are known to be preferred TBW hosts. Modeling predicted that resistance to Bollgard II would not evolve for any natural or current effective scenarios for either the North Carolina or Georgia region.

For the other sampled states, the data more variable both within each growing season and between seasons. In the Delta states ( Mississippi, Louisiana, and Arkansas), the general trend was high proportions of non cotton TBW early in the season, followed by a decline after June ( when cotton became available as a host). During this period, the percentage of cotton origin frequently dipped below 50%, though it was almost always at least 20% ( see table 3). In Texas, during the cotton season ( July October) a majority of TBW were found to be cotton origin, with the proportion exceeding 80% in some individual counties. These results seem to indicate that cotton is a much more important host for TBW in these states then in the east coast states ( North Carolina and Georgia) and that fewer alternate hosts may be available during the cotton season.

BPPD has identified several issues with the TBW sampling and gossypol methodology. First, the use of pheromone traps for sampling resulted in the collection of only males. As such, no  57 females were included in the bioassays. It is unclear whether inferences about female behavior ( i. e. host utilization) can be made from data obtained solely from males. BPPD also questions whether the two year testing period is adequate for an experiment of this magnitude. For both Tennessee and Texas, only one year of data were compiled. Though relatively consistent, cropping and landscape patterns can change over time  a factor that may not be reflected in a study of limited duration. Given the inherent variability in natural and agronomic ecosystems, a two year study may present only a " snapshot" of the host availability and productivity for TBW that may not be representative of future conditions. It is recommended that Monsanto address both of these issues in a future submission.

In addition to the sampling questions, BPPD noted that for a number of counties, trapping locations, and trapping periods there were low trap captures of TBW. For many traps, few ( or no) moths were collected. For example, for Mississippi during July, 2004 ( the height of the cotton season), in half of the sampled counties ( 6 of 12) less than 10 moths were collected. In Tennessee, TBW trap captures were so few that no gossypol analyses were conducted during 2004. Low TBW numbers have also been observed with Bt cotton resistance monitoring efforts ( see BPPD 2005), possibly due to a suppressive effect from the widespread adoption of transgenic cotton. Data from collection sites with few captures ( i. e. < 10) were included in the effective refuge calculations, but not in the modeling ( where worst case scenarios were used). It is unclear if the low trap captures in some areas may have affected the reliability of the results. There is also concern that should a resistant population emerge locally from a Bt cotton field, there will be insufficient susceptible TBW available to mitigate the threat.

Overall, the modeling suggests that Bollgard II cotton should have more than 30 years of durability for the control of TBW in all regions with natural refuge as the only source of susceptible insects. The predictions of the Caprio's spatially explicit, stochastic, two locus TBW resistance model ( Caprio 2006) are similar to those predicted by the Monsanto's three gene, deterministic model ( Gustafson & Head 2005). From a qualitative perspective, modeling indicates the intrinsic durability of Bollgard II is greater than WideStrike which is greater than Bollgard. The presence of larger amounts of Bollgard II in the marketplace also increases the durability of other Bt cotton products that are present but the intrinsic durability of Bollgard II cotton is much greater than Bollgard or WideStrike cotton. However, there are uncertainties in the modeling parameters and assumptions which could impact the modeling output. Modeling suggests that the overall durability of Bollgard II cotton can be enhanced if Bollgard cotton is removed from the marketplace. Uncertainties in the pheromone captures, gossypol analyses, spatial analysis, estimation of effective refuge calculation, degree of shared binding affinity of Cry1Ac to receptor A and B, genetics of resistance, resistance mechanism, initial resistance allele frequency, and other modeling assumptions affect the precision and accuracy of the modeling predictions. Additional competition binding studies using TBW brush border membrane vesicles at saturable concentrations of Cry1Fa are recommended. Uncertainty in the shared binding affinity of the Cry1Ac protein to receptor A and B ( in Monsanto's model) would impact the modeling predictions for all three Bt cotton products because the Cry1Ac protein is common to all of them. Monsanto's modeling also assumes no prior selection to Cry1Ac even  58 though Bollgard cotton has been on the market since 1996. The degree to which the modeling predictions would be impacted is unknown.

It is important to consider that Bollgard II ( Cry1Ac and Cry2Ab2) exists in a mosaic with single gene Bollgard ( Cry1Ac). Modeling work has shown that in such a scenario, the pyramided product can be at risk for resistance if there is significant acreage of a single gene variety ( Zhao et al. 2003, 2005; Hurley 2000). If resistance develops to the single gene product ( Cry1Ac in this case) the dual gene product would effectively become a single gene product ( Cry2Ab2). The single gene variety may in a sense act as a " stepping stone" for resistance to the pyramided product. Zhao et al. ( 2005) have shown that the concurrent use of one and two gene Bt broccoli plants resulted in control failure of both types of Bt plants for control of Plutella xylostella ( diamondback moth) rather than the use of the two gene plants by themselves. In addition, these researchers illustrated through their modeling efforts that resistance will evolve first to the toxin that is being used singly. Monsanto's natural refuge proposal contained no details on how this mosaic might be managed. There was no discussion as to whether Monsanto will " phase out" Bollgard to facilitate the adoption of Bollgard II. Rather, it was suggested in the submission that if there is no requirement to plant structured refuge a strong incentive will exist for growers to switch to Bollgard II. However, considering the large acreage of Bollgard currently in the marketplace ( relative to Bollgard II), it is highly likely that a significant percentage of cotton acreage will remain planted with Bollgard varieties for at least several years.

The potential use of natural refuge for Bollgard II represents a switch from structured refuge ( i. e. refuge planted and managed by growers) to an unstructured ( i. e. unmanaged) refuge. There have been three major considerations to designing and deploying structured refuge for Bt crops. These are 1) production of sufficient numbers of susceptible insects to dilute any potential resistance genes, 2) proximity of the refuge to the Bt field to ensure random mating between susceptible and resistant insects, and 3) synchrony of the refuge with the Bt fields to ensure overlapping emergence between susceptible moths and any resistant survivors of the Bt crop. When evaluating the functionality of natural ( unstructured) refuge, it is useful to consider these topics.

In terms of susceptible production, the goal of structured refuge for Bt crops has historically been a ratio of 500 susceptible insects for every resistant insect that could emerge from the Bt field ( see EPA 2001). Monsanto's TBW sampling/ gossypol experiments did not look at susceptible insect production from a numerical perspective per se; rather the proportion of moths originating on cotton/ non cotton sources was assessed ( all trapped moths can be assumed to be Bt susceptible). The experiments clearly showed that a portion of the TBW develops on cotton, while another portion develops on non cotton crops and wild hosts. However, as discussed above, the numbers collected at individual traps were highly variable, with some traps collecting few or no moths and others trapping over one hundred. It is noted that the sampling was conducted under the current IRM strategy that mandates a non Bt cotton structured refuge which should ( theoretically) have supplied a source of susceptible moths. On the other hand, should the natural refuge strategy be employed, the acreage of Bollgard II cotton could significantly increase while overall non Bt cotton acreage may decrease. Given this scenario, it is conceivable  59 that the portion of TBW developing on cotton ( as determined from Monsanto's experiments) could be reduced, leaving mostly non cotton origin moths. It is not clear whether reducing the cotton origin portion of the susceptible TBW population would have a measurable effect on the ability to dilute emerging resistance genes.

Both refuge proximity and developmental synchrony criteria were directly addressed by Monsanto's experimental design. For proximity, the TBW traps were placed adjacent to cotton fields ( both Bt and non Bt cotton). Though the traps were baited with a pheromone attractant that could attract moths from surrounding areas, the sampling likely presented an accurate portrayal of the TBW population around the cotton field trap sites. Developmental synchrony was also examined by collecting samples throughout the growing season. Data were assessed for each month of the cotton season and a statistical analysis showed that in the majority of cases the cotton/ non cotton proportions did not significantly differ within the individual sampling months. At variable proportions, the results showed that TBW developing on non cotton hosts overlapped TBW from cotton during the major part of the cotton growing season ( July September) ( see figure 1 attached to this review). These results were not surprising: though TBW may develop on different plant hosts at slightly different rates, there are four to six generations per year that likely create a natural overlap ( discussion in Benedict 2004).

BPPD generally agrees with Monsanto that a natural refuge strategy ( allowing 100% Bollgard II) will result in economic and environmental benefits. These benefits will result largely from yield increases and reduced insecticide use on cotton acreage previously planted as refuge ( i. e. conventional cotton). However, these benefits will likely be compromised should the lepidopteran target pests develop resistance to the Bollgard II toxins. In such a case, there could be significant costs to growers that might have to switch to conventional ( insecticide treated) cotton.

Under the current terms and conditions of registration for approved Bt cotton products, registrants are required to monitor for pest resistance to the expressed toxins. To date, BPPD has reviewed the annual monitoring reports through the 2004 growing season and found no evidence of resistance to Cry1Ac or Cry2Ab2 in either TBW or CBW ( see BPPD 2005). Should the natural refuge proposal ultimately be accepted, it is highly recommended that these monitoring efforts continue for both Cry1Ac and Cry2Ab2. Monsanto's submission did not discuss any proposed changes to the monitoring requirements for either Bollgard product. However, considering that replacement of structured refuge with natural refuge may increase the Bollgard II acreage ( and selection pressure for resistance), it may be justified to increase the monitoring efforts for Cry2Ab2 ( i. e. increased sampling sites and collections).

References Monsanto Submissions ( others referenced by author in the next section)

MRID# 455457 01: Insect Resistance Management Plan for Bollgard II Cotton. Submitted in 2002.  60 MRID# 467172 01: " Production of Tobacco Budworm from Alternate Host Plants and the Role of These Host Plants as Natural Refuge for Bollgard II Cotton." Submitted December 20, 2005.

MRID# 467172 02: " Modeling the Impact of Natural Refuge on the Evolution of Tobacco Budworm and Cotton Bollworm Resistance to Bollgard II Cotton." Submitted December 20, 2005.

MRID# 467172 03: " Scientific and Economic Justification for Not Requiring Structured Cotton Refuges for Bollgard II Cotton in the U. S. Cotton Belt from Texas to the East Coast." Submitted December 20, 2005.

April 3, 2006: Letter to Dennis Szuhay containing supplemental information requested in support of natural refuge amendment request for Bollgard II cotton. Supplemental to MRID# 467172 01.

April 7, 2006: Letter to Dennis Szuhay containing supplemental information requested in support of natural refuge amendment request for Bollgard II cotton. Supplemental to MRID# 467172 02.

May 10, 2006: Letter to Dennis Szuhay containing supplemental information requested in support of natural refuge amendment request for Bollgard II cotton. Supplemental to MRID# 467172 02.

Author References Abney, M. R., C. E. Sorenson and J. J. R. Bradley, 2004. Alternate crop hosts as resistance management refuges for tobacco budworm in NC. Pages 1413 1416 in Proceedings of the Beltwide Cotton Conferences, San Antonio, Texas. National Cotton Council.

Banerjee, S. and S. W. Martin, 2005. Farm profits from observed yields of Bt and non Bt cotton, with and without spray applications, in the Mississippi Delta. Proceeding of the Beltwide Cotton Conference, New Orleans, Louisiana, p. 297 303.

Benedict, J. H., 2004. Biology and Dispersal of the Cotton Bollworm and Tobacco Budworm in North America ( white paper). Submitted in Monsanto's unpublished study " Production of Tobacco Budworm from Alternate Host Plants and the Role of These Host Plants as Natural Refuge for Bollgard II Cotton." MRID# 467172 01.

Bird, L. J. and R. J. Akhurst, 2004. Relative fitness of Cry1A resistant and  susceptible Helicoverpa armigera ( Lepidoptera: Noctuidae) on conventional and transgenic cotton. J Econ Entomol 97( 5): 1699 1709.

Blanco, C. A., 2005. Bacillus thuringiensis Cry1Ac/ Cry2Ab2 resistance monitoring program for  61 tobacco budworm and bollworm in 2004. Unpublished study submitted to EPA. MRID # 465476 01.

Blanco, C. A., L. C. Adams, J. Gore, D. Hardee, M. Mullen, J. R. Bradley, J. W. Van Duyn, P. C. Ellsworth, J. K. Greene, D. R. Johnson, R. Luttrell, G. Studebaker, A. Herbert, M. Karner, R. Leonard, B. Lewis, J. J. D. Lopez, D. Parker, M. L. Williams, R. Parker, M. E. Roof, R. Sprenkel, S. D. Stewart, J. R. Weeks, S. Carroll, M. Parajulee, P. Roberts and J. Ruberson, 2004. Bacillus thuringiensis monitoring program for tobacco budworm and bollworm in 2003. Pages 1327 1331 in Proceedings of the Beltwide Cotton Conferences, San Antonio, Texas. National Cotton Council.

Burd, A. D., 2001. The influence of environmental factors on susceptibility of B. t. cottons to bollworm, Helicoverpa zea, and factors affecting resistance to B. t. toxins for bollworm. Raleigh, North Carolina State University.

Burd, A. D., J. R. Bradley, Jr., J. W. Van Duyn, and F. Gould, 2001. Estimated frequency of nonrecessive B. t. resistance genes in bollworm, Helicoverpa zea. Proceedings of the Beltwide Cotton Conferences. Vol 2: 820 822.

Candas, M., O. Loseva, B. Oppert, P. Kosaraju, and L. A. Bulla, 2003. Insect resistance to Bacillus thuringiensis  alterations in the Indianmeal moth larval gut proteome. Mol. Cell. Proteomics 2: 19 28.

Caprio, M. A., 1998a. Two compartment model for insect resistance. http:// www. msstate. edu/ Entomology/ PGjava/ ILSImodel. html [ December 1, 2005].

Caprio, M. A., 1998b. Evaluating resistance management strategies for multiple toxins in the presence of external refuges. J Econ Entomol 91( 5): 1021 1031.

Caprio, M. A., 2006. Draft Report: Modeling the risk of resistance in Heliothis virescens to Bollgard II cotton. Provided by Monsanto Company, April 7, 2006.

Caprio, M. A., M. K. Faver and G. Hankins, 2004. Evaluating the impacts of refuge width on source sink dynamics between transgenic and non transgenic cotton. J Insect Sci 4( 3): 1 5.

Carrière, Y., C. Ellers Kirk, Y. B. Liu, M. A. Sims, A. L. Patin, T. J. Dennehy and B. E. Tabashnik, 2001a. Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm ( Lepidoptera: Gelechiidae). J Econ Entomol 94( 6): 1571 1576.

Carrière, Y., C. Ellers Kirk, A. L. Patin, M. A. Sims, S. Meyer, Y. b. Liu, T. J. Dennehy and B. E. Tabashnik, 2001b. Overwintering cost associated with resistance to transgenic cotton in the pink bollworm ( Lepidoptera: Gelechiidae). J Econ Entomol 94( 4): 935 941.  62 Carrière, Y., C. Ellers Kirk, R. Biggs, D. M. Higginson, T. J. Dennehy and B. E. Tabashnik, 2004. Effects of gossypol on fitness costs associated with resistance to Bt cotton in pink bollworm. J Econ Entomol 97( 5): 1710 1718.

Carrière, Y., C. Ellers Kirk, R. Biggs, B. Degain, D. Holley, C. Yafuso, P. Evans, T. J. Dennehy and B. E. Tabashnik, 2005. Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm ( Lepidoptera: Gelechiidae) to Bt cotton. J Econ Entomol 98( 3): 947 954.

Constable, G. A., D. J. Llewellyn and P. E. Reid, 1998. Biotechnology risks and benefits: the Ingard ® cotton example. http:// www. csu. edu. au/ special/ agronomy/ papers [ December 1, 2005].

Doane Agricultural Services, 2004. U. S. farm seed corn study report.

Ferré, J. and J. VanRie, 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu. Rev. Entomol 47: 501 533.

Gould, F., 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu Rev Entomol 43( 1): 701 726.

Gould, F., A. Anderson, A. Jones, D. Sumerford, D. G. Heckel, J. Lopez, S. Micinski, R. Leonard and M. Laster, 1997. Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens. PNAS 94( 8): 3519 3523.

Gould, F., N. Blair, M. Reid, T. L. Rennie, J. Lopez and S. Micinski, 2002. Bacillus thuringiensis toxin resistance management: stable isotope assessment of alternate host use by Helicoverpa zea. PNAS 99( 26): 16581 16586.

Greenplate, J. T., 2004. Report on studies to assess supplemental pyrethroid spray effects on Helicoverpa zea populations in Bollgard ® cotton. Monsanto Company, Report number MSL 19252, MRID 46222402. St. Louis, Missouri.

Greenplate, J. T., J. W. Mullins, S. R. Penn, A. Dahm, B. J. Reich, J. A. Osborn, P. R. Rahn, L. Ruschke and Z. W. Shappley, 2003. Partial characterization of cotton plants expressing two toxin proteins from Bacillus thuringiensis: relative toxin contribution, toxin interaction, and resistance management. J Appl Entomol 127: 340 347.

Gunning, R. V., H. T. Dang, F. C. Kemp, I. C. Nicholson, and G. D. Moores, 2005. New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis. Appl. and Env. Microbio. 71: 2558 2563.

Gustafson, D. and G. Head, 2004. Impact of updated alternative host and pyrethroid effectivness data on model predictions of cotton bollworm resistance development to Bollgard ® cotton. Monsanto Company, Report number MSL 19326. St. Louis, Missiouri.  63 Gustafson, D., G. Head and M. A. Caprio, 2005. Modeling the impact of alternative hosts and pyrethroid sprays on Helicoverpa zea ( Boddie) adaptation to Bollgard ® cotton. J Econ Entomol. ( submitted).

Gustafson, D., G. Head and H. K. Reding, 2001. Impact of effective refuge size and typical insecticide use practices on model predictions of years to resistance of tobacco budworm and cotton bollworm to Bollgard ® cotton. Monsanto Company, Report number MSL 19229, MRID# 462224 03. St. Louis, Missouri.

Head, G., R. E. Jackson, J. R. Bradley, J. W. Van Duyn, B. R. Leonard, R. Luttrell, J. Ruberson, J. J. Adamczyk, J. Gore, D. Hardee, R. Voth, S. Sivasupramaniam and J. W. Mullins, in draft. Regional host utilization by Helicoverpa zea as measured by stable carbon isotope analyses.

Head, G. and R. Voth, 2004. A final report on studies to assess production of Helicoverpa zea from alternate host plants and from the external unsprayed non Bt cotton refuge for Bollgard ® cotton. Monsanto Company, Report number MSL 19238, MRID 46222401. St. Louis, Missouri.

Hurley, T. M. 2000. Research report: A Bioceconomic evluation of the gradual introduction of multiple toxin Bt corn. Prepared for Monsanto Company. February 2000.

Jackson, R. E., J. R. Bradley, F. Gould and J. W. Van Duyn, 2003. B. t. resistance evolution in the Helicoverpa zea population in eastern North Carolina. Pages 1168 1176 in Proceedings of the Beltwide Cotton Conferences, Nashville, Tennessee. National Cotton Council.

Jackson, R. E., J. R. Bradley and J. W. Van Duyn. 2005a. Comparative efficacy of Bt technologies against bollworm in North Carolina. Pages 1373 1378 in Proceedings of the Beltwide Cotton Conferences, New Orleans, Louisiana. National Cotton Council.

Jackson, R. E., J. R. Bradley, J. W. Van Duyn, B. R. Leonard, R. Luttrell, J. Ruberson, J. J. Adamczyk, J. Gore, D. Hardee, R. Voth, S. Sivasupramaniam, J. W. Mullins and G. Head, 2005b. Regional assessment of Helicoverpa zea ( Lepidoptera: Noctuidae) populations on cotton and non cotton crop hosts. J Econ Entomol ( submitted).

Jurat Fuentes, J. L. and M. L. Adang, 2001. Importance of Cry1   endotoxin domain II loops for binding specificity in Heliothis virescens ( L.). Appl. and Env. Entomol. 67: 323 329.

Jurat Fuentes, J. L., F. L. Gould, and M. J. Adang, 2003. Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance. Appl Environ Microbiol 69 ( 10): 5898 5906.

Koziel, M. G., G. L. Beland, C. Bowman, N. B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Lewis, D. Maddox, K. McPherson, M. R. Meghji, E. Merlink, R. Rhodes, G. W. Warren, M. Wright and S. V. Evola, 1993. Field performace of an elite  64 transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Biotechnology 11: 194 200.

Kurtz, R. W., F. Gould and J. J. R. Bradley, 2004. Helicoverpa zea fitness on Bt corn and cotton in eastern North Carolina: potential effects of alternate host crops and pyramided Bt plants. Pages 1430 1434 in Proceedings of the Beltwide Cotton Conferences, San Antonio, Texas. National Cotton Council.

Livingston, M. J., G. A. Carlson and P. L. Fackler, 2004. Managing resistance evolution in two pests to two toxins with refugia. Am J Agr Econ 86: 1 13.

Luttrell, R., L. Wan and K. Knighten, 1999. Variation in susceptibility of noctuid ( Lepidoptera) larvae attacking cotton and soybean to purified endotoxin proteins and commercial formulations of Bacillus thuringiensis. J Econ Entomol 92: 21 32.

Mahon, R., K. Olsen, S. Young, K. Garsia, L. Lawrence, 2004. Resistance to Bt toxins in Helicoverpa armigera. The Australian Cotton Grower. December 2003 January 2004. p. 8 10.

Martin, S. W., K. Bryant, K. Paxton, and S. Banerjee, 2006. Preliminary evaluation of Bt refuges based on on farm observations. Submitted as poster for 2006 Beltwide Cotton Conferences, Memphis, Tennessee.

Onstad, D. W., D. W. Crowder, P. D. Mitchell, C. A. Guse, J. L. Spencer, E. Levine and M. E. Gray, 2003. Economics versus alleles: balancing integrated pest management and insect resistance management for rotation resistant western corn rootworm ( Coleoptera: Chrysomelidae). J Econ Entomol 96( 6): 1872 1885.

Orth, R. G., G. Head, and M. Mierkowski, in draft. Determining larval host plant use by a polyphagous lepidopteran through analysis of adult moths for plant secondary metabolites.

Perlak, F. J., M. Oppenhuizen, K. Gustafson, R. Voth, S. Sivasupramaniam, D. Heering, B. Carey, R. A. Ihrig and J. K. Roberts, 2001. Development and commercial use of Bollgard ® cotton in the USA  early promises versus today's reality. The Plant Journal 27( 6): 489 501.

Rausher, M. D., 2001. Co evolution and plant resistance to natural enemies. Nature 411( 6839): 857 864.

Roush, R. T., 1994. Managing pests and their resistance to Bacillus thuringiensis: can transgenic crops be better than sprays? Biocontrol Sci Techn 4: 501 516.

Roush, R. T., 1998. Two toxin strategies for management of insecticidal transgenic crops: can pyramiding succeed where pesticide mixtures have not? Philos T Roy Soc 353: 1777 1786.

Schneider, J. C., 2003. Overwintering of Heliothis virescens ( F.) and Helicoverpa zea ( Boddie)  65 ( Lepidoptera: Noctuidae) in Cotton Fields of Northeast Mississippi. J Econ Entomol 96 ( 5): 1433 1447.

Tabashnik, B. E., Y. Carrière, T. J. Dennehy, S. Morin, M. S. Sisterson, R. T. Roush, A. M. Shelton and J. Z. Zhao, 2003. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Econ Entomol 96( 4): 1031 1038.

Tabashnik, B. E., F. Gould and Y. Carriere, 2004. Delaying evolution of insect resistance to transgenic crops by decreasing dominance and heritability. J Evolution Biol 17( 4): 904 912.

USDA NASS, 2005. Quick Stats. United States Department of Agriculture. http:// www. nass. usda. gov/ QuickStats/ [ December 1, 2005].

Whitten, M. J., R. A. Jefferson and D. Dall, 1996. Biotechnology and integrated pest management in Biotechnology in Agriculture Series. 15. CAB International, Washington, D. C.

Zhao, J. Z., J. Cao, Y. Li, H. L. Collins, R. T. Roush, E. D. Earle, and A. M. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology Online: http:// www. nature. com/ naturebiotechnoloy .

Zhao, J. Z., J. Cao, H. L. Collins, S. L. Bates, R. T. Roush, E. D. Earle and A. M. Shelton, 2005. Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants. PNAS. 102: 8426 8430.

EPA References BPPD, 2002a. EPA Review of Monsanto Company's Bollgard II Cotton Insect Resistance Management Plan for Section 3 Full Commercial Registration. S. Matten memorandum to L. Cole, October 24, 2002.

BPPD, 2002b. EPA Review of Benefits for Monsanto Company's Bollgard II Cotton For Section 3 Full Commercial Registration. E. Brandt/ S. Matten/ A. Reynolds memorandum to L. Cole, November 6, 2002.

BPPD, 2004a. Technical review of Monsanto's submission: " A Final Report on Studies to Assess Production of Helicoverpa zea from Alternate Host Plants and from the External Unsprayed Non Bt Cotton Refuge for Bollgard ® Cotton." S. Matten memorandum to L. Cole, April 22, 2004.

BPPD, 2004b. Technical review of Monsanto's submission: " Impact of Effective refuge size and typical insecticide use practices on model predictions of years to resistance of tobacco budworm and cotton bollworm to Bollgard cotton." S. Matten memorandum to L. Cole dated April 22, 2004.  66 BPPD, 2005. Review of 2004 monitoring data submitted by Monsanto for Bt cotton ( Bollgard and Bollgard II). A. Reynolds memorandum to L. Cole, December 1, 2005.

EPA, 1998. Bacillus thuringiensis ( B. t.) plant pesticides and resistance managment. United States Environmental Protection Agency, Report number EPA 738 F 98 001.

EPA, 2001. Bt plant incorporated protectants October 15, 2001 biopesticides registration action document. Available at http:// www. epa. gov/ pesticides/ biopesticides.

SAP, 2004. Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting, June 8 10, 2004. A Set of Scientific Issues Being Considered by the U. S. Environmental Protection Agency Regarding: Product Characterization, Human Health Risk, Ecological Risk, and Insect Resistance Management for Bacillus thuringiensis ( Bt) Cotton Products. United States Environmental Protection Agency, Washington, D. C. http:// www. epa. gov/ scipoly/ sap/ 2004/ june/ final1a. pdf.