### C h a r g e q u e s t i o n s
8

. D o e s t h e P a n e l h a v e a n y r e c o m m e n d a t io n s f o r d is t r ib u t in g n o n m u lt ip lic a t iv e in t e r a c t io n s in m o d e ls t o e v a lu a t e m u lt i- g e n e p y r a m id e d p r o d u c t s ?

9

. P le a s e c o m m e n t o n t h e r e lia b ilit y o f t h e e s t im a t e s o f r e s is t a n c e e v o lu t io n b y e a c h o f t h e t h r e e m o d e ls in lig h t o f t h e b io lo g ic a l a n d p a r a m e t e r u n c e r t a in t ie s id e n t ifi e d b y B P P D

.

## Modeling
S e e d b l e n d s v s

. S t r u c t u r e d r e f u g e
D i f f e r e n c e s
L a r v a l m o v e m e n t ( b l e n d s ) A d u l t d i s p e r s a l ( r e f u g e s )

## Models
M o n s a n t o - i m p l i c i t e v e r y t h i n g
D o w - e x p l i c i t l y s p a t i a l
E P A / O R D

- c o m p a r t m e n t , i m p l i c i t l y s p a t i a l , f r e q u e n c y - o n l y
I v e s e t a l

. ( 2 0 1 0 )

- s p a t i a l l y i m p l i c i t a n d e x p l i c i t , d e n s i t y - d e p e n d e n t o r f r e q u e n c y - o n l y

### G o a l : U n d e r s t a n d p r o c e s s e s t h a t a f f e c t r a t e o f r e s is t a n c e e v o lu t io n
M a k e c o m p a r is o n s a m o n g s c e n a r io s :

“ A ll e ls e e q u a l, ” w h a t is t h e b e s t s t r a t e g y ? Given different models and target insects, need to understand differing results Estimates of time to resistance failure are inherently suspect (even if r esults from 3 models are similar)
It is easier to assess whether o ne scenario leads to faster or slower resistance evolution than predicting when failure will occurSS SR RR SS SR RRSurvival 1

0Survival 1

0 Faster evolution Survival on Bt plants (not realistic values) Higher survival

Lower survival Heuristic e xplanation of high-dose strategySS SR RRSurvival 1

0

SS SR RRSurvival 1

0 Slower evolution (due to relative survival of S R to SS) Add refuge Higher survival

Lower survival Survival at population level (not realistic values)

Information provided on SS survival Contrast between data and what models u se/needResults preview

1

. I f a d u l t s h a v e h i g h d i s p e r s a l a m o n g f i e l d s a n d l a r v a e d o n o t m o v e a m o n g p l a n t s , t h e n b l e n d s a n d r e f u g e s a r e e q u i v a l e n t

. 2

. S t r u c t u r e d r e f u g e s : L i m i t e d a d u l t d i s p e r s a l c a n s p e e d b u t m a i n l y s l o w s r e s i s t a n c e e v o l u t i o n ( d e p e n d i n g o n m a l e v s

. f e m a l e d i s p e r s a l r a t e s , r o t a t i o n p a t t e r n s , e t c

. )

. 3

. B l e n d s : F o r h i g h

- d o s e c a s e , l a r v a l m o v e m e n t a m o n g p l a n t s w i l l m o s t l y s p e e d r e s i s t a n c e e v o l u t i o n , w i t h a n i m p o r t a n t r o l e o f i n c r e a s e d s u r v i v a l o f h e t e r o z y g o t e s

. 4

. E x p r e s s i o n o f r e s i s t a n c e i n h e t e r o z y g o t e s i s k e y t o t h e d u r a b i l i t y o f s e e d b l e n d s , y e t k e y i n f o r m a t i o n i s n o t k n o w n

. 5

. M o n s a n t o , D o w , a n d E P A / O R D m o d e l s f o r t h e d u r a b i l i t y o f 5 % s e e d b l e n d s a r e N O T c o n s e r v a t i v e , e s p e c i a l l y f o r S W C B
I w i l l p r e s e n t e x a m p l e s h i g h l i g h t i n g p r o c e s s e s t h a t a r e n o t a d e q u a t e l y a d d r e s s e d i n a n y o f t h e m o d e l s

.General Model
Ives, A. R., P. R. Glaum, N. L. Ziebarth, and D. A. Andow. 2 0 1 0

. The evolution of resistance to two-toxin pyramid transgenic crops. Ecological Applications i n press
S h a r e s c h a r a c t e r i s t i c s w i t h o t h e r m o d e l s ( n o t i n d e p e n d e n t )
2 B t t o x i n s ( C R W ) t h a t a c t i n d e p e n d e n t l y
2 p a r t i a l l y r e c e s s i v e r e s i s t a n c e a l l e l e s
R a n d o m m a t i n g w i t h i n f i e l d s
P r e - m a t i n g m a l e d i s p e r s a l , p o s t

- m a t i n g f e m a l e d i s p e r s a l
K e e p t r a c k o f b o t h d e n s i t y a n d a l l e l e f r e q u e n c i e s
E i t h e r d e n s i t y - d e p e n d e n t s u r v i v a l o f l a r v a e o n p l a n t s , o r n o t ( f r e q u e n c y - o n l y m o d e l = e x t i n c t i o n m o d e l )
F o r m o f d e n s i t y d e p e n d e n c e i r r e l e v a n t

### 1 0

. 0 6 0

. 0 1 R R 0

. 0 6 0

. 0 0 3 5 0

. 0 0 0 6 S R 0

. 0 1 0

. 0 0 0 6 0

. 0 0 0 1 S S R R S R S S 

## S u r v i v a l s o n B t p l a n t s

Relative survival of heterozygotes, h = 0.05

s X X Y Y = s X X s Y Y Different from EPA/ORD, but will return to this1. I f adults h ave high dispersal among f ields and larvae do not move among plants, then blends and refuges are equivalent.2. Structured refuges: Limited adult dispersal can speed or slow resistance evolution, depending on male vs. female dispersal rates, rotation patterns, etc. Two opposing forces: Limited dispersal means insects remain in refuges (provided incomplete rotation) slows resistance Limited dispersal can cause resistance hotspots speeds resistance
I will give a brief description only to allay fears about structured refuges.Moderate a dult dispersal

w h i t e = a r e a s o f h i g h e r a l l e l e f r e q u e n c y g e n e r a t i o n 9 1 g e n e r a t i o n 9 2Low adult dispersal

w h i t e = a r e a s o f h i g h e r a l l e l e f r e q u e n c y g e n e r a t i o n 3 2 g e n e r a t i o n 3 3Assumptions 5% refuge fields exponential dispersal kernel (including some adults remaining in natal fields) pre-mating male dispersal, post-mating female dispersal refuge rotation either every 3 generations, or never

### R e f u g e r o t a t io n e v e r y 3 g e n e r a t io n s

I n c l u d e s c o n t r a s t i n g e f f e c t s o f ( i ) t h e p r o b a b i l i t y o f l e a v i n g n a t a l f i e l d s a n d ( i i ) t h e d i s t a n c e d i s p e r s e d o n c e n a t a l f i e l d s a r e l e f t ( A n d o w , C h a r g e Q u e s t i o n 1 )

### N o r e f u g e r o t a t io n2. Structured refuges: Limited adult dispersal can speed or slow resistance evolution, depending on male vs. female dispersal rates, rotation patterns, etc. Limited dispersal will generally slow rather than speed resistance evolution.

B u t extremely limited dispersal can greatly speed resistance evolution.3. Blends: For high-dose case, larval movement among plants will mainly speed resistance evolution, with an important role of increased survival of heterozygotes.
Unknowns:
Behavior: m ovement rates, response to density, stage at movement, response to Bt vs. non-Bt plants, mortality during moves Susceptibility: p re-movement selection, postmovement selection, survival of heterozygotes to multiple toxins Studies need to target unknown information Onstad et al. comment expands on unknowns3. Blends: For high-dose case, larval movement among plants will mainly speed resistance evolution, with an important role of increased survival of heterozygotes.
Unknowns:
Behavior: m ovement rates, response to density, stage at movement , response to Bt vs. non-Bt plants, mortality during moves Susceptibility: p re-movement selection, postmovement selection, survival of heterozygotes to multiple toxins Studies need to target unknown information Onstad et al. comment expands on unknowns1 0.24 0.1 RR 0.24 0.06 0.024 SR 0.1 0.024 0.01 SS RR SR SS 

### S e e d b le n d s : s u r v iv a l o f la r v a l s t a g e s Pre- and post-movement stages Assume fixed probability of moving between stages (same for Bt and non-Bt plants; independent of density) Move randomly to other plants

s X X Y Y = s X X Y Y 1 s X X Y Y 2 (like EPA/ORD, but not on a daily time scale) Stage-specific survivalsLarval movement = probability of leaving initial plant

SSSS survival includes survival on non-Bt plants

Relative RS survivalincludes survival on nonBt plants1 0.1051 0.058 

### R R 0.0861 0.0233 0.02 

### S R 0.038 0.019 0.018 

### S S R R S R S S E P A / O R D S u r v iv a ls ( lo w - d o s e , C R W )

1 0.06 0.01 

### R R 0.06 0.0035 0.0006 

### S R 0.01 0.0006 0.0001 

### S S R R S R S S S u r v iv a ls u s e d h e r e ( h ig h d o s e , 2

- t o x in E C B )Larval movement has almost no effect f or lowdose crops

Relative R S survival unaffected w ith low-dose cropsDoes the timing of larval movement (stage of strongest selection) affect resistance evolution?

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 0.06 0.01 R R 0.06 0.0035 0.0006 S R 0.01 0.0006 0.0001 S S R R S R S S

1 0.06 0.01 R R 0.06 0.0035 0.0006 S R 0.01 0.0006 0.0001 S S R R S R S S 1 1 1 R R 1 1 1 S R 1 1 1 S S R R S R S S

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 1 1 R R 1 1 1 S R 1 1 1 S S R R S R S S Stage 1 Stage 2 Case 1 Case 3 Case 2Does the timing of larval movement (stage of strongest selection) affect resistance evolution?

When there is pre-movement selection (case 2), movement slows resistance evolution Removes density-dependent mortality of stage 1 susceptibles on non-Bt plants C a s e 1 = s p l i t 2 = s t a g e 1 s e l e c t i o n 3 = s t a g e 2 s e l e c t i o nDoes the timing of larval movement (stage of strongest selection) affect resistance evolution?

C a s e 1 = s p l i t 2 = s t a g e 1 s e l e c t i o n 3 = s t a g e 2 s e l e c t i o nDoes the timing of larval movement (stage of strongest selection) affect resistance evolution?

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 0.5 0.31 R R 0.5 0.24 0.16 S R 0.31 0.16 0.1 S S R R S R S S

1 0.5 0.31 R R 0.5 0.24 0.16 S R 0.31 0.16 0.1 S S R R S R S S 1 0.12 0.032 R R 0.12 0.015 0.004 S R 0.032 0.004 0.001 S S R R S R S S

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 0.12 0.032 R R 0.12 0.015 0.004 S R 0.032 0.004 0.001 S S R R S R S S Stage 1 Stage 2 Case 1 Case 5 Case 4Does the timing of larval movement (stage of strongest selection) affect resistance evolution?Summary In moderate/high dose cases, larval movement speeds resistance evolution, but not in low-dose cases SAP for Pioneer ’ s Optimum AcreMax 1 recommended 20% seed mix To my knowledge, there has been no systematicstudy of the consequences of selection preversus post-movement Most importantly, we need to know the strength ofselection on heterozygotes, both pre- and postmovement

### C h a r g e q u e s t i o n
8

. D o e s t h e P a n e l h a v e a n y r e c o m m e n d a t io n s f o r d is t r ib u t in g n o n m u lt ip lic a t iv e in t e r a c t io n s in m o d e ls t o e v a lu a t e m u lt i- g e n e p y r a m id e d p r o d u c t s ?

### E P A / O R D ( w it h C r y 1 F = S S )

0

. 0 0 5 0 2 0

. 0 0 1 7 6 0

. 0 0 1 4 0 

### R R 0

. 0 0 1 7 6 0

. 0 0 1 4 4 0

. 0 0 1 4 0 

### S R 0

. 0 0 1 4 0 0

. 0 0 1 4 0 0

. 0 0 1 4 0 

### S S R R S R S S

0

. 0 9 9 7 6 0

. 0 0 1 9 0 Cry2Ab2 0

. 1 1 5 0 0 0

. 0 0 2 7 5 Cry1A.105 Dominance Survivorship of SS0

. 0 0 5 0 2 0

. 0 0 2 7 7 0

. 0 0 2 6 5 

### R R 0

. 0 0 2 7 7 0

. 0 0 1 5 3 0

. 0 0 1 4 6 

### S R 0

. 0 0 2 6 5 0

. 0 0 1 4 6 0

. 0 0 1 4 0 

### S S R R S R S S E P A / O R D ( w it h C r y 1 F = S S )

0

. 0 0 5 0 2 0

. 0 0 1 7 6 0

. 0 0 1 4 0 

### R R 0

. 0 0 1 7 6 0

. 0 0 1 4 4 0

. 0 0 1 4 0 

### S R 0

. 0 0 1 4 0 0

. 0 0 1 4 0 0

. 0 0 1 4 0 

### S S R R S R S S

W it h h 1 

### = h 2 

### = 0

. 0 5

### I n c r e a s e s u r v iv a ls b y 5 x

1 0.06 0.01 

### R R 0.06 0.0035 0.0006 

### S R 0.01 0.0006 0.0001 

### S S R R S R S S

S a m e s u r v iv a ls p r e - a n d p o s t

m o v e m e n t

R S R S = c r o s s r e s is t a n c e5x survival of SRSS o r SRSR halves the time to resistance failure1 0.06 0.01 

### R R 0.06 0.0035 0.0006 

### S R 0.01 0.0006 0.0001 

### S S R R S R S S I n c r e a s e s u r v iv a ls b y 5 x

1 0.06 0.01 

### R R 0.06 0.0035 0.0006 

### S R 0.01 0.0006 0.0001 

### S S R R S R S S

A s y m m e t r ic v s

. S y m m e t r icAsymmetric

Symmetric

### D if f e r e n c e s b e t w e e n lo c i in r a t e o f e v o lu t io n Rate of resistance is driven largely by the locus that evolves most rapidly

c ompare three cases:

s A , S S = s B , S S = 0.01

s A , S S = 0.01, s B , S S = 0.001

s A , S S = s B , S S = 0.001Summary

Estimates ( guesses) of r elative survivals of SRSS and SRSR genotypes are poor They have a large impact on the rate of resistance evolution Asymmetric changes in survivals of heterozygotes appear to h ave e ffects almost as large as symmetric changes
Overall rate of resistance evolution seems to be driven by the fastest evolving locus

### C h a r g e q u e s t i o n
8

. D o e s t h e P a n e l h a v e a n y r e c o m m e n d a t io n s f o r d is t r ib u t in g n o n m u lt ip lic a t iv e in t e r a c t io n s in m o d e ls t o e v a lu a t e m u lt i- g e n e p y r a m id e d p r o d u c t s ?

W e n e e d t o u s e m o d e ls t o e x a m in e w h a t g e n e in t e r a c t io n s w ill m o s t lik e ly g iv e t h e m o s t r a p id r e s is t a n c e e v o lu t io n ( s e n s it iv it y a n a ly s e s a r e lim it e d )
U lt im a t e ly , h o w g e n e s in e a c h t a r g e t s p e c ie s in t e r a c t is a n e m p ir ic a l q u e s t io n

### C h a r g e q u e s t i o n s
9

. P le a s e c o m m e n t o n t h e r e lia b ilit y o f t h e e s t im a t e s o f r e s is t a n c e e v o lu t io n b y e a c h o f t h e t h r e e m o d e ls in lig h t o f t h e b io lo g ic a l a n d p a r a m e t e r u n c e r t a in t ie s id e n t ifi e d b y B P P D

.Monsanto model When larvae move, a key effect is on the relative survival of heterozygotes
For ECB and SWCB, Monsanto models movement of larvae implicitly as a decrease in refuge size to account for increased larval mortality This largely misses the pointMonsanto model underestimates the importance of larval movement and overestimates the time to resistance failure.Dow model Models CRW with high-dose parameters which generally cause slower resistance evolution than low-dose parameters
Nonetheless, u nder low-dose conditions, larval movement l ikely makes l ittle difference, so seed blends are less of an issue for CRW Only investigated change in allele frequencies over 20 years, so results are hard to assessEPA/ORD model Does not include density - “ e xtinction model ” Density-dependence is important:

L a r v a l m o r t a l i t y d e c r e a s e s s u r v i v a l o f s u s c e p t i b l e s
I t t h e r e b y i n c r e a s e s t h e r e l a t i v e s u r v i v a l o f m o r e r e s i s t a n t h e t e r o z y g o t e s , a n d s p e e d s r e s i s t a n c e e v o l u t i o n (ii) For ECB and SWCB, apparently l ittle increased survival of heterozygotes (iii) issues involving sensitivity analysisFrequency-only and density-dependent models coincide when populations are going extinct (and resistance evolution is rapid)5. M onsanto, Dow, and EPA/ORD models f or the durability o f 5% s eed blends are NOT conservative, especially for SWCB

Monsanto - larval movement had no effect on relative survival of heterozygotes Dow - used high-dose survivals for CRW (as pointed out by BPPD)
EPA/ORD - ( i) no density-dependent larval survival; (ii) for ECB and SWCB, apparently little increased survival of heterozygotes; (iii) issues involving sensitivity analysisWhat are the relative survivals of SRSS, SSSR, and SRSR heterozygotes? What are the relative strengths of selection on pre- vs. post-movement larvae? These questions cannot be answered with theory

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 0.5 0.31 R R 0.5 0.24 0.16 S R 0.31 0.16 0.1 S S R R S R S S

1 0.5 0.31 R R 0.5 0.24 0.16 S R 0.31 0.16 0.1 S S R R S R S S 1 0.12 0.032 R R 0.12 0.015 0.004 S R 0.032 0.004 0.001 S S R R S R S S

1 0.24 0.1 R R 0.24 0.06 0.024 S R 0.1 0.024 0.01 S S R R S R S S

1 0.12 0.032 R R 0.12 0.015 0.004 S R 0.032 0.004 0.001 S S R R S R S S Stage 1 Stage 2 Case 1 Case 5 Case 4

### C h a r g e q u e s t i o n s
9

. P le a s e c o m m e n t o n t h e r e lia b ilit y o f t h e e s t im a t e s o f r e s is t a n c e e v o lu t io n b y e a c h o f t h e t h r e e m o d e ls in lig h t o f t h e b io lo g ic a l a n d p a r a m e t e r u n c e r t a in t ie s id e n t ifi e d b y B P P D

. H a r d t o s a y H a t e t o g u e s s I w o u ld n ’ t p la c e m o n e y o n t h e m

### C o n c l u s i o n s
1

. S t r u c t u r e d r e f u g e s a r e b e t t e r t h a n s e e d b le n d s f o r E C B a n d S W C B

I ssues of compliance and variation in blended seed clustering not addressed


### 2

. M o n s a n t o , D o w , a n d E P A / O R D m o d e ls f o r t h e d u r a b ilit y o f 5 % se e d b le n d s a r e N O T c o n s e r v a t iv e , e s p e c ia lly f o r S W C B

### I n f o r m a t i o n t h a t i s l a c k i n g
1

. C E W n e e d s t o b e a d d r e s s e d w it h d a t a a n d in t e r - r e g io n a l m o d e ls
2

. S u r v iv a l o f h e t e r o z y g o t e s , e p it a s is , a n d h o w t h e y in t e r a c t w it h la r v a l m o v e m e n t f o r E C B a n d e s p e c ia lly S W C B ( n e e d f o r a “ m o d e l s y s t e m ” )
3

. Q u a n t it a t iv e g e n e t ic m o d e ls f o r lo w d o s e p r o d u c t s
4

. M o n it o r in g / r e m e d ia t io n s t r a t e g ie s f o r s t a c k e d p r o d u c t s

### H o w r e lia b le a r e t h e e s t im a t e s o f t im e t o f a ilu r e f o r t h e M o n s a n t o , D o w , a n d E P A / O R D m o d e ls ?
N ic k w o u ld s a y N O T
G r a h a m s a id t h a t t h e a b s e n c e o f c a s e s o f r e s is t a n c e m e a n s t h a t t h e m o d e ls h a v e e it h e r b e e n r ig h t o r t o o c o n s e r v a t iv e
B u t w h a t a b o u t H e lic o v e r

p a p u n c t

ig e r

a in A u s t r a lia ? T h is is a m o n g t h e la s t p e s t s y o u w o u ld e x p e c t t o e v o lv e r e s is t a n c e

.Helicoverpa punctigera

Resistance developing to cry2Ab in Bollgard II Downes, S., T. Parker, and R. Mahon. 2 0 1 0 . Incipient resistance of H elicoverpa punctigera to the Cry2 Ab Bt toxin in Bollgard II (R) cotton. PloS ONE 5 (9 ).

b u t F 1 s c r e e n w a s s i g n i f i c a n t l y l o w e r i n 2 0 0 9 / 1 0

2 0 0 9 /1 0Helicoverpa punctigera

Resistance developing to cry2Ab in Bollgard II Downes, S., T. Parker, and R. Mahon. 2 0 1 0 . Incipient resistance of H elicoverpa punctigera to the Cry2 Ab Bt toxin in Bollgard II (R) cotton. PloS ONE 5 (9 ).

b u t F 1 s c r e e n w a s s i g n i f i c a n t l y l o w e r i n 2 0 0 9 / 1 0

2 0 0 9 /1 0

Do we have enough information to model resistance accurately?

### R e c o m m e n d a t i o n
E P A s h o u ld c o n s id e r in t h e ir r is k a s s e s s m e n t a b r o a d e r r a n g e o f I R M t a c t ic s For example:
1. Introduce 20% SmartStax seed b lend
2. Monitor insect population M o n s a n t o / D o w d a t a d e m o n s t r a t e s u r v i v a l o f s u s c e p t i b l e s a t t h e fi e l d s c a l e , b u t w e n e e d i n f o r m a t i o n a t t h e r e g i o n a l s c a l e 3. In 5 years, if insect populations are not too low, consider a 10% seed b lend 4. In 10 years, consider a 5% seed b lend