Sampling and Methodology
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The Panel
is asked to comment on the pheromone sampling strategy employed by Monsanto in
which only male tobacco budworm (TBW) were trapped.
Is this an appropriate sampling
strategy? Can inferences about female TBW be derived from data gathered
exclusively with males?
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Monsanto’s TBW sampling and gossypol analyses were conducted over a two year period (2004
and 2005). For several states (Tennessee and E. Texas) data were collected in
only one year. The trends between seasons were generally consistent, although
no statistical/correlation analysis was performed.
The Panel is asked to comment on what
uncertainties exist from using data collected from this time period (i.e., 2
years for North Carolina and Georgia and 1 year for Tennessee and E. Texas) to
adequately assess the potential of natural refuge (i.e., non-cotton hosts) as a
substitute for structured refuge (i.e., non-Bt cotton)?
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In
some counties/states, extremely low numbers of TBW were trapped, with some
traps collecting only one insect. In Tennessee, TBW numbers were so low that
data were not reported at all for 2004. In addition, cotton monitoring efforts
have been recently hampered by low availability of TBW samples (possibly due to
a suppressive effect of Bt cotton).
Do low overall numbers of TBW trap
captures in some areas affect the ability to assess the effectiveness of
natural refuge for IRM? What conclusions, if any, should be drawn from the
failure to capture Bt-susceptible TBW at particular sites?
Statistical Analyses
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Monsanto
used the Fisher’s Exact Test to determine whether the gossypol data could be
pooled. Data were pooled for individual traps (i.e. for multiple collection dates
for each month) and for counties (i.e. including all traps within a county for
each month).
The Panel is asked to comment on Monsanto’s approach to pooling the gossypol data.
- Monsanto did not conduct any statistical analyses comparing the two sampling years (2004
and 2005). The Panel is asked to comment on whether valid comparisons (on a
qualitative basis) can be made between the two years without statistical analyses?
Please describe any meta-statistical analysis that could improve the overall understanding
of the effectiveness of natural refuge across locations and across time.
Effective Refuge Calculation
and Modeling
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Monsanto
has corrected their calculation of effective refuge size presented in Gustafson
and Head, 2004 based on the Agency’s (BPPD, 2004b) and June 2004’s Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel’s
(SAP) recommendations (SAP, 2004). Modifications to the calculation of the
effective refuge size involved removing the assumption of constant effective
refuge size and explicitly accounting for the lower production of CBW and TBW
in cotton where survival of these insects is reduced. Estimation of the
effective refuge now assumes a regionally specific annual cycle of effective
refuge size, according to data collected in alternative host studies of CBW (Head and Voth, 2004) and TBW (Head and Gustafson, 2005). These data were combined with corn planting estimates on either the regional scale for CBW, or county-scale for TBW, to estimate effective (i.e., current (structured non-Bt cotton + non-cotton) and natural (non-cotton only) refuge sizes for each of what were conservatively assumed to be six annual generations for each pest.
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Estimation of the relative number of CBW adult moths produced by each of the five
sub-compartments is given by the following equation: Mij
= Aij Eij LBij LSij (Equation
1).
[M is the number of adult moths produced per unit area of the region; A is
the proportion of the region occupied by the crop type of interest; E is
the relative (to cotton, i.e. Ecotton=1) number of effective
eggs (eggs that would produce adults in the absence of B.t. or
pyrethroid sprays) laid in the crop type; LB is the fraction of larvae
surviving in the presence of the B.t. crop; LS is the fraction of
larvae surviving a pyrethroid insecticide spray on the crop; the subscript i
refers to the compartment (B for B.t. or R for refuge);
and the subscript j refers to the particular crop type within the
compartment (1 = cotton, 2 = corn, 3 = other C3 host crop).]
The effective refuge, Reff, is defined as the proportion of
adult moths that would have been produced in the refuge compartment (non-Bt
cotton, non-Bt corn, non-cotton C3 crops) in the absence of any induced larval
mortality:
Reff = (Equation
5; used when CBW populations were actively feedings in cotton, Generations
3-5)
Effective refuge estimations for
all of the “non-cotton” generations are given by:
= (Equation
6)
The natural refuge component
(i.e., non-cotton C3 crops + non-Bt corn components) of the total effective
refuge is as follows:
= (Equation
7)
The Agency asks the SAP to
comment on the estimated CBW effective and natural refuge calculations.
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Pooled, county-level estimates of the percent cotton-reared TBW moths
were combined with county-level landcover information to estimate the current
effective refuge and natural refuge for each county per month. The relative
TBW productivity of non-cotton areas within a county for a specific month is
given as:
ENC = (Equation
8)
The current effective refuge
(non-Bt cotton + non-cotton hosts) 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 estimated natural refuge
(non-cotton hosts) for TBW is given by the following equation:
= (Equation
10)
The Agency asks the SAP to
comment on the estimated TBW effective and natural refuge calculations.
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Monsanto
examined the durability of each of the three Bt cotton products (i.e.,
Bollgard, WideStrike, and Bollgard II) individually and together in the
marketplace using a three-gene model. The Bt protein, Cry1Ac, is common to all
three products. The presence of each of these products in the marketplace
selects for potential resistance to Bollgard cotton, expressing only the Cry1Ac
protein, and also selects for resistance to the other two products through the
common selection for Cry1Ac resistance. The products vary greatly in the rate
at which they select for resistance to Cry1Ac because of the presence of
additional insecticidal proteins in Bollgard II (Cry2Ab2) and in WideStrike (Cry1F).
The three-gene model for insect
resistance evolution used in this study is based on a conceptual model similar
to that proposed by Dow AgroSciences (DAS) for its
product, WideStrike cotton, and was reviewed by a recent U.S. EPA Scientific
Advisory Panel (SAP) (SAP, 2004). However, the SAP questioned some of the mathematical details of the DAS model and Monsanto has made some changes to address the SAP’s concerns. As shown in schematic form in Figure 3, Appendix 2, the three-gene model is based on the following assumptions concerning the mechanism of activity of the three commercial Bt cotton products (Bollgard, Bollgard II, and WideStrike cotton):
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The Cry1Ac toxin, present in all three products, binds to two
receptors, 60% to receptor A and 40% to receptor B.
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The Cry1F toxin, present only in WideStrike cotton, binds
exclusively to receptor A.
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The Cry2Ab2 toxin, present only in Bollgard II cotton, binds
exclusively to receptor C.
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CBW. Based
both on the intrinsic durability of each of the three B.t. cotton
products (Figure 4, Appendix 2) and the three-gene modeling analyses for
all three Bt cotton products together in the marketplace (Table 14, Appendix
2), Bollgard II retained the highest level of efficacy against CBW in all
scenarios (all regions). 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, Appendix 2). This is because of the relatively high
mortality of individuals heterozygous to Cry1Ac resistance in the presence of
Cry2Ab2, as compared to WideStrike. WideStrike is intermediate in many
scenarios because of the shared binding receptor between Cry1F and CryAc and the
likelihood of cross-resistance is greater. Bollgard is weakest in all
scenarios because there is no high dose for CBW and it is 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 to
all three Bt cotton products in the North Carolina region in all scenarios,
even the natural refuge scenarios. When Bollgard cotton acreage is minimized,
Bollgard II and WideStrike longevity is maximized (Table 14). Large
amounts of Bollgard II cotton in the marketplace increased the durability of
both Bollgard and WideStrike (Table 14, Appendix 2). Uncertainties in
the pheromone captures, estimation of adult productivity, carbon isotope
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. Monsanto’s modeling also does not consider pre-selection for
Cry1Ac resistance. Ten years of selection pressure (since 1996) for resistance
to Cry1Ac has already occurred. Field resistance to Cry1Ac places additional
selection pressure on the Cry2Ab2 component of Bollgard II cotton.
Given the assumptions and
uncertainties in Monsanto’s CBW modeling efforts, the Agency asks the SAP to
comment on the utility of the modeling to predict the effectiveness of natural
(non-cotton C3 crops + non-Bt corn) vs. current effective refuge (non-Bt cotton
+ non-Bt corn + non-cotton C3 crops) to manage CBW resistance to the toxins
expressed in Bollgard II. Discuss the impact of pre-selection for Cry1Ac
resistance on the modeling output.
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TBW. 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). 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. 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, Appendix 2), 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, 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. Monsanto’s modeling also does not consider
pre-selection for Cry1Ac resistance. Ten years of selection pressure (since
1996) for resistance to Cry1Ac has already occurred. Field resistance to
Cry1Ac places additional selection pressure on the Cry2Ab2 component of
Bollgard II cotton.
Given the assumptions and
uncertainties in Monsanto’s TBW modeling efforts, the Agency asks the SAP to
comment on the utility of the modeling to predict the effectiveness of natural
(non-cotton hosts) vs. current effective refuge (non-Bt cotton + non-cotton
hosts) to manage TBW resistance to the Bt toxins expressed in Bollgard II
cotton. Discuss the impact of pre-selection for Cry1Ac resistance on the
modeling output.
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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,
2005). 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. Bollgard, Widestrike, and Bollgard II cotton exist in a mosaic in
southeastern cotton growing regions, with Bollgard dominating the total
acreage. In 2004, Bollgard cotton acreage accounted for >95% of all Bt cotton
acreage in the U.S. (see Head et al., 2005, MRID# 467172-03). 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 would benefit the two-gene products, Bollgard II and
WideStrike.
The Panel is asked to address the
implications for selection for CBW and TBW resistance if the mosaic of single
gene and dual gene products remains in the marketplace for a number of years. How
would selection pressure be reduced if the single gene product is removed from
the marketplace gradually (e.g., >3 years) or rapidly (e.g., ≤3 years)
over a period of years?
Overall Data/Results
Interpretation
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There
are three major variables to evaluating structured refuge for Bt crops: a)
production of a sufficient number of susceptible insects relative to any
resistant survivors of the Bt crop, b) proximity of the refuge to the
transgenic crop to facilitate random mating between susceptible (from the
refuge) and resistant (from the Bt crop) insects, and c) developmental
synchrony of the refuge with the transgenic crop to promote random mating.
Given Monsanto’s sampling,
gossypol analysis, spatial and temporal analyses, and modeling evaluation, the
Agency asks the panel to comment on whether Monsanto’s analysis scientifically
supports the conclusion that natural refuge will be comparable to the
effectiveness of structured refuge for management of TBW resistance to the Bt proteins
expressed in Bollgard II cotton for each of the four regions: the Carolinas,
Georgia, Mississippi Delta, and Texas.
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