PRODUCTION AND CHARACTERIZATION OF BT CRY1AC RESISTANCE IN 
BOLLWORM, HELICOVERPA ZEA (BODDIE) 
 
 
Except where reference is made to the work of others, the work described in this 
dissertation is my own or was done in collaboration with my advisory committee.  
This dissertation does not include proprietary or classified information. 
 
 
 
 
Konasale Jayaramu Anilkumar 
 
 
Certificate of approval: 
 
 
 
     
Arthur G. Appel  William J. Moar, Chair 
Professor  Professor 
Entomology and Plant Pathology  Entomology and Plant Pathology 
   
   
     
Nannan Liu  Sakuntala Sivasupramaniam 
Associate Professor  Senior Research Scientist 
Entomology and Plant Pathology  Monsanto Company, St. Louis, MO 
   
   
     
James T. Bradley  George T. Flowers 
Professor  Interim Dean 
Biological Sciences  Graduate School  
 
PRODUCTION AND CHARACTERIZATION OF BT CRY1AC RESISTANCE IN 
BOLLWORM, HELICOVERPA ZEA (BODDIE) 
 
 
 
Konasale Jayaramu Anilkumar 
 
 
 
 
 
A Dissertation  
Submitted to 
the Graduate Faculty of 
Auburn University 
in Partial Fulfillment of the  
Requirements for the  
Degree of  
Doctor of Philosophy 
 
 
 
 
 
 
 
 
 
Auburn, Alabama 
August 9, 2008 
iii 
 
PRODUCTION AND CHARACTERIZATION OF BT CRY1AC RESISTANCE IN 
BOLLWORM, HELICOVERPA ZEA (BODDIE) 
 
 
 
Konasale Jayaramu Anilkumar 
 
 
 
 
Permission is granted to Auburn University to make copies of this dissertation 
at its discretion, upon request of individuals or institutions at their expense. 
The author reserves all publication rights. 
 
 
 
 
 
Signature of Author 
 
 
 
Date of Graduation 
 
 
iv 
 
DISSERTATION ABSTRACT 
PRODUCTION AND CHARACTERIZATION OF BT CRY1AC RESISTANCE IN 
BOLLWORM, HELICOVERPA ZEA (BODDIE) 
 
 
 
Konasale Jayaramu Anilkumar 
 
Doctor of philosophy, August 9, 2008 
(M.Sc. University of Agricultural Sciences, Bangalore, India, 2002) 
(B.Sc. University of Agricultural Sciences, Bangalore, India, 2000) 
 
201 Typed Pages 
 
Directed by William J. Moar 
 
 
Laboratory-selected Bt-resistant colonies are important tools for elucidating Bt 
resistance mechanisms and helping to determine appropriate resistance management 
strategies for Bt crops.  Here, two laboratory populations of Helicoverpa zea (AR and 
MR), resistant to Bt Cry1Ac, were established by selection with either Cry1Ac activated 
toxin (AR) or MVP II (MR) from an unselected parent strain (SC).  Stable and high level 
resistance was achieved in AR but not in MR.  AR was only partially cross-resistant to 
MVP II suggesting that MVP II does not have the same Cry1Ac selection pressure as 
Cry1Ac toxin against H. zea and that proteases may be involved with resistance.  AR was 
highly cross-resistant to Cry1Ab toxin.  AR was not cross-resistant to
 
v 
 
Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A and cypermethrin.  Toxin 
binding assays showed no significant differences, indicating that resistance was not 
linked to a reduction in binding.  
In response to selection, heritability values for AR increased in generations 4 to 7 
and decreased in generations 11 to 19.  While rearing on Cry1Ac treated diet, AR had 
significantly increased pupal mortality, a male-biased sex ratio, and lower mating success 
compared to SC.  AR males had significantly more mating costs compared to females.  
AR had significantly higher fitness costs in involving larval mortality, weight, and 
period; pupal weight, period, and mortality compared to SC.  Cry1Ac-resistance was not 
stable in AR in the absence of selection.   
In laboratory experiments with field-cultivated Bt and non-Bt cotton squares AR 
significantly outperformed SC.  However, AR could not complete larval development on 
Bt cotton. Additionally, a significantly lower percentage of AR larvae reached pupation 
on non-Bt compared with SC.  Diet incorporation bioassays indicated Cry1Ac was 
significantly more lethal to SC compared to AR; however, no differential susceptibility 
was observed in strains for gossypol.  Combinations of Cry1Ac with gossypol, cotton and 
corn powder were synergistic against AR, but not against SC.   These results may help 
understand the inability of AR to complete development on Bt cotton. 
These results 1) highlight the need to choose carefully the form of Bt protein used 
in experimental studies, 2) support the lack of success of selecting, and maintaining 
Cry1Ac-resistant populations of H. zea in the laboratory, and 3) aid in understanding why 
this major pest of cotton and corn has not yet evolved Bt resistance. 
vi 
 
ACKNOWLEDGMENTS 
 
 
It has been a great pleasure working with faculty, staff and students at the Auburn 
University, during my tenure as a doctoral student; I thank all of them for their help, 
suggestions and camaraderie.  I greatly acknowledge efforts of William J. Moar, my 
adviser for helping me to develop as a scientist/good citizen through his guidance, 
motivation and insight.  I sincerely thank other members of my committee, Arthur Appel, 
Nannan Liu, James Bradley, and Sakuntala Sivasupramaniam and my outside reader, 
Mark Liles, for their time and genuine interest.  My sincere thanks to Edzard van Santen 
for providing insight and helping me in statistical analysis.  I thank Moar?s lab members 
Nathan Burkett, Megan Leach, Emily Boydson, and Jayadevi Chandrashekar for helping 
me in taking care of my insects while I was away from lab.  I am grateful to my parents 
Jayaramu and Sowbhagya who helped me see this through from start to finish, to whom I 
dedicate this dissertation.  I especially thank my good friend, Pratap Simha, and other 
friends at Auburn and elsewhere for several years of laughter, camaraderie, and 
stimulating, if not always intellectual, conversation.  Finally, I profoundly thank my wife, 
Thara, for her unfailing love, her gentle but persistent encouragement, her kindness and 
her patience kept me sane and happy all the while. 
vii 
 
Style manual of journal used: Chapter 1, Journal of Economic Entomology; 
Chapter 2, Applied and Environmental Microbiology; Chapter 3, Journal of Economic 
Entomology; Chapter 4, Journal of Economic Entomology  
Computer software used: Microsoft
?
 office 2003, 2007, SAS for Windows
?
 
version 9.1, Sigma Plot
?
 version 10.0, SPSS version 14.0 
 
viii 
 
TABLE OF CONTENTS 
 
 
LIST OF TABLES ............................................................................................................. ix 
LIST OF FIGURES ........................................................................................................... xi 
CHAPTER 1:  THE RESEARCH INTRODUCTION, OBJECTIVES AND 
LITERATURE REVIEW ................................................................1 
CHAPTER 2:  PRODUCTION AND CHARACATERIZATION OF BT  
CRY1AC-RESISTANCE IN COTTON BOLLWORM, 
HELICOVERPA ZEA (BODDIE) ................................................81 
CHAPTER 3:  FITNESS COSTS ASSOCIATED WITH CRY1AC-RESISTANT  
HELICOVERPA ZEA (LEPIDOPTERA: NOCTUIDAE): A  
FACTOR COUNTERING SELECTION FOR RESISTANCE TO  
BT COTTON ...............................................................................116 
CHAPTER 4: SYNERGISTIC INTERACTIONS BETWEEN CRY1AC AND 
GOSSYPOL LIMIT SURVIVAL OF CRY1AC-RESISTANT 
HELICOVERPA ZEA (LEPIDOPTERA: NOCTUIDAE) ON BT 
COTTON .....................................................................................152 
 
ix 
 
LIST OF TABLES 
 
CHAPTER 1 
Table 1. Selected insect species and strains that have developed resistance to Bt 
proteins .........................................................................................................................65 
CHAPTER 2 
Table 1. Resistance development in H. zea when selected using activated toxin and 
MVP II .......................................................................................................................106 
Table 2. Cross-resistance of AR to MVP II ...............................................................108 
Table 3. Cross-resistance of AR to other B. thuringiensis proteins and  
Cypermethrin .............................................................................................................109 
Table 4. Dissociation constants (K
d
) and concentration of binding sites (R
t
) for 
binding of Cry1A proteins to BBMV from H. zea. ...................................................110 
CHAPTER 3 
Table 1. Heritability (h
2
) and resistance risk assessment for resistance to Cry1Ac in 
H. zea .........................................................................................................................140   
Table 2. Reproductive propensity (mean ? SE) of Cry1Ac-resistant (AR) and 
susceptible (SC) strains of H. zea during selection and rearing, respectively ...........141 
Table 3. The reproductive (mean ? SE) success in a Cry1Ac-resistant (AR), 
susceptible (SC) and their reciprocal crosses  ............................................................142 
x 
 
Table 4. Fitness parameters (mean ? SE) for Cry1Ac-resistant (AR) and susceptible 
(SC) strains of H. zea .................................................................................................143 
Table 5. Growth rate (mean ? SE) for Cry1Ac-resistant (AR) and susceptible (SC) 
strains of H. zea on different strengths of diet ...........................................................144 
Table 6.  Fitness parameters (mean ? SE) for Cry1Ac-resistant (AR1) and susceptible 
(SC1) strains of H. zea after crossing AR with SC1 ..................................................145 
CHAPTER 4 
Table 1. Lethal time (LT) to mortality for a Cry1Ac-resistant (AR) and a susceptible 
(SC) strains on Bt (DPL555) and NBt (DPL491) cotton squares ..............................180 
Table 2. Performance of a Cry1Ac-resistant (AR) and a susceptible (SC) H. zea on Bt 
(DPL-555) and NBt (DPL-491) cotton squares. ........................................................181 
Table 3. Molt inhibitory concentration (failure to molt to third instar) response of a 
Cry1Ac-resistant (AR1) and a susceptible (SC1) H. zea to Cry1Ac, and its 1:1 
mixture with gossypol. ...............................................................................................182 
Table 4. Weight stunting concentration response of a Cry1Ac-resistant (AR1) and a 
susceptible (SC1) strain of H. zea to Cry1Ac, gossypol and their 1:1 mixtures. .......183 
Table 5. Percent mortality of Cry1Ac-resistant (AR1) and a susceptible (SC1) H. zea 
in 4% cotton/corn powder in the presence and absence of 15 ?gCry1Ac/g diet. ......184 
Table 6. Interactions of Cry1Ac with gossypol and cotton powder as measured by 
failure to molt into third instars after seven days. ......................................................185 
 
xi 
 
LIST OF FIGURES  
 
CHAPTER 2 
Figure 1. Toxicity of Cry1Ab (a) and Cry2Ab2 (b) expressing corn leaf powder to 
susceptible (SC) and Cry1Ac-resistant (AR) H. zea. .................................................111 
 
Figure 2. Binding of 
125
I-Cry1Ac (A) and 
125
I-Cry1Aa (B) to BBMV from susceptible 
(SC) (z) and Cry1Ac-resistant (AR) H. zea (S) at increasing concentrations of 
unlabeled homologous competitor .............................................................................112 
Figure 3. Percent binding of 
125
I-Cry1Ac to BBMV from susceptible (SC) and 
Cry1Ac-resistant (AR) H. zea in the absence (SC and AR) or the presence (LC+GN 
and AR+GN) of 25 mM GalNac. ...............................................................................113 
CHAPTER 3 
Figure 1. Pupal sex ratio of AR and SC strains over time with selection and rearing, 
respectively ................................................................................................................146 
Figure 2. Mating success in AR and SC over time with selection and rearing, 
respectively ................................................................................................................147 
Figure 3. Growth rate differences in AR and SC, when larvae were reared on regular 
diet after exposing to regular diet and selection diet (20% diluted regular diet) for 
initial seven days ........................................................................................................148 
xii 
 
Figure 4. Percent survivors of Cry1Ac-resistant H. zea at 500 ?g/g Cry1Ac toxin 
when selected continuously at 500 ?g/g Cry1Ac toxin compared to when removed 
from selection for one generation ..............................................................................149 
CHAPTER 4 
Fig 1. Quantity of Cry1Ac protein (?g Cry1Ac/g lyophilized tissue) expressed in Bt 
cotton squares using ELISA.......................................................................................186 
Fig 2. Cumulative % mortality of susceptible (SC) and Cry1Ac-resistant (AR) H. zea 
on Bt (DPL-555) and NBt (DPL-491) cotton squares. ..............................................187 
Fig 3. Concentration of gossypol in Cry1Ac-resistant (AR1) and a susceptible (SC1) 
H. zea. ........................................................................................................................188 
Fig 4. Effect of 4% cotton/corn powder in the presence and absence of 15 ?g 
Cry1Ac/g of diet on larval weight in Cry1Ac-resistant (AR1) and a susceptible (SC1) 
H. zea. ........................................................................................................................189 
 
 
1 
CHAPTER 1:  
THE RESEARCH INTRODUCTION, OBJECTIVES AND REVIEW OF 
LITERATURE 
 
1.1 Introduction  
       Currently registered insect-protected plants are genetically transformed to express 
insecticidal proteins from Bacillus thuringiensis and are referred to as Bt crops. Since 
their commercial introduction into the US in 1996, growers have rapidly adopted Bt crops 
as an effective tool to increase yield by effectively controlling insect pests. Bt crops 
constitutively produce insecticidal toxins throughout the life of the plant providing 
excellent control of primary target insects and are a very important component of 
integrated pest management practices (Shelton et al. 2002).  
  
Bt cotton (Bollgard
?
) was the first large scale commercialized Bt transgenic crop 
in the US (Perlak et al. 1990) and has been cultivated on approximately 52% (2.8 m Ha) 
of the total US cotton acreage in 2006 (Brookes and Barfoot 2006). The adoption of Bt 
cotton has helped US farmers increase their income by $50/Ha and reduce insecticide use 
by 841.5 metric tons of insecticides in 2001 (James 2002).  However, the success of Bt 
cotton may be short lived if the target pest(s) develop resistance due to the widespread 
crop plantings and prolonged exposure to Bt toxins. Concerns regarding the development 
of Bt resistance by the target pests are so great that an insect resistance management 
2 
(IRM) plan is mandated by EPA (Environmental Protection Agency) as part of the 
registration package for Bollgard
?
 as well as other Bt crops (Bates et al. 2005).   
 
In the US, Bollgard
? 
expresses Bt Cry1Ac protoxin, and is cultivated primarily to 
control tobacco budworm (TBW) Heliothis virescens (F.) pink bollworm (PBW) 
Pectinophora gossypiella (Saunders), and, to a lesser extent, cotton bollworm (CBW) 
Helicoverpa zea (Boddie).  Many TBW and PBW strains derived from laboratory 
selection have demonstrated their ability to adapt to Cry1Ac. Results from these studies 
have contributed greatly to insect resistant management (IRM) policy making discussed 
above that have, arguably, helped to delay Bt resistance development in cotton (Gould 
1998, 2000).  Interestingly, studies with laboratory-selected resistant strains of these two 
species have shown that resistance characteristics and mechanisms are related to both 
toxin and species (Tabashnik et al. 2003b).  The TBW resistant strain YHD2 with 
10,100-fold resistance to Cry1Ac failed to survive, but PBW resistant strain AZP-R with 
little over 3,000-fold resistance to Cry1Ac could complete its development on Bt cotton, 
even though both species have similar susceptibilities to Cry1Ac (MacIntosh et al. 1990, 
Tabashnik et al. 2003b, Sivasupramaniam et al. 2008).   Although Bt resistance was at 
least partly due to an altered cadherin-like protein for both species,  the mutation in TBW 
lead to a single amino acid change (Xie et al. 2005) while an 8 amino acid deletion 
occurred in PBW  (Morin et al. 2003).  
 
Although TBW and PBW have been extensively studied with regards to Bt 
resistance, similar studies have not been conducted with CBW even though it has a 
naturally higher tolerance (Stone and Sims 1993, Luttrell et al. 1999, Sivasupramaniam et 
3 
al. 2008) to Cry1Ac than the other two target species, which can result in occasional 
completion of larval development on Bollgard
?
 (Jackson et al. 2004). This increased 
tolerance increases the likelihood that resistance could evolve. Similar concerns have also 
been expressed in relation to Helicoverpa armigera (H?bner), the primary target pest of 
Bt cotton in the Old World and validated with laboratory selection experiments. H. 
armigera (Akhurst et al. 2003) is known to have similar susceptibility (LC
50
=10?g/g) to 
Cry1Ac as H. zea (MacIntosh et al. 1990) and studies conducted with Cry1Ac-Sel (13-
fold resistance, Fan et al. 2000) and BX strain (57-fold resistance, Akhurst et al. 2003) 
showed 25 and 58% survival on Bt cotton, respectively. Resistance in H. armigera is 
inherited as a partial recessive character (Bird and Akhurst 2004, Kranthi et al. 2006) 
contrary to the observed recessive inheritance in PBW and TBW (Tabashnik et al. 2004). 
Hence, it remains to be seen as to how CBW responds to Cry1Ac-selection, the 
inheritance of developed resistance and the fitness costs, if any, associated with the 
evolution of resistance. 
 
Selection experiments cited above on PBW, TBW and H. armigera have been 
conducted with either MVP II or Cry1Ac crystals (containing protoxin). Because 
Bollgard
? 
expresses Bt Cry1Ac solubilized protoxin that is at least partly activated to 
toxin within cotton tissue (Gao et al. 2006), we hypothesize that selection experiments 
using MVP II or Cry1Ac crystals may not adequately reflect resistance selection that is 
taking place in planta. Several physiological processes (solubilization and proteolysis) 
must occur before the Bt protein present in Bt protoxin is toxic to insects, and at least one 
of these processes (proteolysis) has been documented to be associated with Bt resistance 
4 
(Oppert et al. 1997).  Therefore, we proposed to conduct selection experiments, in 
parallel with both MVP II as well as Cry1Ac toxin. Even though selection using 
Bollgard
?
 tissues would be more realistic, proprietary research restrictions with 
Bollgard
?
 limited our scope of research. 
1.2 The research goal and specific objectives 
The present study has been undertaken with the following objectives. 1) To select 
for resistance to the Cry1Ac protein using MVP II and activated Cry1Ac toxin; 2) To 
characterize the biochemical and molecular mechanisms of resistance such as alteration 
in binding, and altered proteolysis; 3) To ascertain the possible fitness costs associated 
with resistance development; 4) To document cross resistance to other Bt Cry proteins 
and other relevant insecticides belonging to different classes/groups; 5) To study the 
survivorship of resistant and susceptible strains on Bt and non-Bt cotton squares; and 6) 
To investigate the interactions of Cry1Ac and plant secondary metabolites such as 
gossypol on resistant and susceptible straisn. 
1.3 Review of literature 
Reviews of literature pertaining to cotton bollworm, Helicoverpa zea (Boddie), Bt toxins, 
and Bt resistance in various insects has been reviewed and presented. 
 
1.3.1 Cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) 
1.3.1.1 Biology  
H. zea is known to be highly polyphagous and is an economic pest of crops such as corn 
(corn ear worm), cotton (cotton bollworm), tomato (tomato fruit worm), pepper, bean, 
eggplant, alfalfa, sorghum, and soybean (Bergvinson 2005).  
 
5 
Eggs are deposited singly on leaves, fruiting structures, and corn silk.  The shape 
varies from slightly dome-shaped to a flattened sphere, and measures about 0.5 to 0.6 mm 
in diameter and 0.5 mm in height. Eggs are creamy white in color when laid and turn 
black before hatching, after 3-4 days of incubation at room temperature (25-27 
0
C) 
(Ellsworth and Bradley 1992).  
Larval duration is about 15 D with 5-7 instars at room temperature (25-27 
0
C). 
Larvae feed on flower buds, fruits, bolls or pods (Gore et al. 2003). Larval color ranges 
from green, brown and red, to black depending on their host and genetic makeup, and 
have distinct, longitudinal stripes running down the body (Archer and Bynum 1994).  
Larval exoskeleton is roughened by numerous minute spines which is an identifying 
character to distinguish it from H.  virescens larvae (Bailey et al. 2001). First instar larvae 
are not cannibalistic, and therefore, several larvae may feed together initially. However, 
as larvae mature they become very aggressive, killing and cannibalizing other larvae 
(Kolodny-Hirsch and Harrison 1982, Ellsworth and Bradley 1992, Archer and Bynum 
1994, 1998). 
Mature larvae leave the feeding site and drop to the ground, where they burrow 
into the soil and pupate. The larva prepares a pupal chamber 5 to 10 cm below the soil 
surface and the duration of the pupal period is about 7 - 10 days (Butler 1976). 
 
The forewings of the moths usually are yellowish brown in color, and often bear a 
small dark spot centrally. The small dark spot is especially distinct when viewed from 
below. The forewing also may bear a broad dark transverse band distally, but the margin 
of the wing is not darkened. The hind wings are creamy white basally and blackish 
6 
distally, and usually bear a small dark spot centrally. The adult longevity is about a week. 
The sex ratio is 1:1.2 (male: female) and fecundity is about 500-2000 eggs per female 
(Geraud et al. 1996).  
 
The bollworm/budworm complex was the top insect pests of cotton in 2004 
damaging 1.23% of the 2004 US crop.  Almost 82% of crop was infested with the 
complex of which 94% were bollworms (Source: M.R. Williams, 2004. 
www.msstate.edu/Entomology/Cotton.html).  Interestingly, the primary reason bollworm 
is the current predominant lepidopteran ?pest? is due to the high adoption of Bt cotton 
that severely suppressess the budworm population. The heliothine complex probably 
costs cotton farmers worldwide approximately $3 billion annually; which includes yield 
loss and control costs, excluding labor costs for sprays (James 2002).  
 
1.3.1.2 Distribution 
H. zea is widely distributed in southern Canada, Mexico and USA in North America; and 
Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Falkland Islands, French Guiana, 
Guyana, Peru, Surinam, Uruguay, and Venezuela in South America. It is active 
throughout the year in tropical and sub-tropical climates; however, it is restricted to 
summer months in higher latitudes (Bergvinson 2005).  
 
1.3.1.3 Management of H. zea  
Before the advent of Bt cotton H. zea was treated primarily with synthetic insecticides 
belonging to most major classes of insecticides such as carbaryl, esfenvalerate, 
permethrin, spinosad, cyhalothrin, cyfluthrin, bifenthrin, zeta-cypermethrin, methomyl, 
lambda-cyhalothrin, and thiodicarb (Luttrell et al. 1994, Casida and Quistad 1998). 
7 
Widespread insecticide resistance to permethrin, methomyl (Hsu and Yu 1991), 
cyhalothrin (Brown et al. 1997, 1998), cypermethrin (Brown et al. 1997) and other 
insecticides (Leeper and Raffa 1986, Leonard et al. 1988, McCutchen et al.1989, Adb-
Elghafar et al. 1993, Ernst and Dittrich 1992) and rising concerns regarding 
environmental hazards have urged scientists to search for alternatives to conventional 
insecticides (Gould 1991).  As a result of rigorous research exploration, biological and 
microbial insecticides namely Bt (Ali and Young 1996, Lambert et al. 1996), and nuclear 
polyhedrosis virus (NPV) (Bell and Hayes 1994), were commercialized. Although these 
insecticides are eco-friendly and effective, they were short lived because of both biotic 
and abiotic environmental factors. These factors, plus the difficulties faced in the control 
of these pests which feed internally have led to ingeneous research resulting in the advent 
of the insect-protected plants in 1996 (Bergvinson 2005). These insect-protected plants 
are transformed to express insecticidal proteins derived from a soil bacterium, Bacillus 
thuringiensis Berliner (de Maagd et al. 1999a). These plants express the insecticidal 
toxins in all tissues and throughout its life under the influence of the constitutive 
promoters (Cannon 2000). 
 
1.3.2 Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) 
B. thuringiensis (Bt) is a rod shaped aerobic, spore-forming common soil bacterium. Bt 
was first identified in 1901 by Ishiwata as a pathogen of silk worm which causes ?sotto? 
disease and hence it was named Bacillus sotto (Ishiwata 1901, Federici 2005). Later, it 
was isolated from dead Mediterranean flour moth larvae Ephestia kunhniella Keller in 
1911 and named as Bacillus thuringiensis (after Thuringia, Germany) by Berliner 
8 
(Berliner 1915). Bt produces an array of insecticidal compounds belonging to different 
families such as crystal (Cry) proteins (delta-endotoxins), cytolytic proteins, vegetative 
insecticidal proteins, beta-exotoxin, zwittermycin, spore, etc. (de Maagd et al. 2003). 
Among these insecticidal compounds, the most widely studied group are delta-endotoxins 
(Cry proteins).  
 
Cry proteins (Schnepf 1995) are produced during sporulation and are often toxic 
to insects (H?fte and Whiteley 1989, de Maagd et al. 2003). The first recorded Bt trials 
for insect control were conducted in Hungary in the 1920?s and Yugoslavia in the early 
1930?s to control the European corn borer, Ostrinia nubilalis (H?bner) (Beegle and 
Yamamoto 1992). The success of these trials led to the first commercial formulation by 
Laboratoire Libec in France. The product, Sporeine, was available in 1938 (Lord 2005) 
and later in other parts of the world as spray formulations for managing insect pests 
(Beegle and Yamamoto 1992, Tabashnik 1994).  These insecticides were 
environmentally safe and effective due to their high specificity and unique mode of 
action (Lambert and Peferoen 1992, Federici 2005). 
 
Bt
 
is the most diverse species in the B. cereus group (B. thuringiensis, Bacillus 
cereus, Bacillus anthracis, and Bacillus
 
mycoides, as well as the recently described 
Bacillus pseudomycoides and Bacillus weihenstephanensis), and its strains have been 
classified into
 
84 serovars (serovarieties), with over 800 strains (Lecadet et al. 1999, and 
Reyes-Ramirez and Ibarra 2005).  The current nomenclature, classification, and range of 
toxicity to different insects can be found on the Bt Toxin Nomenclature webpage at 
http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ 
9 
 
1.3.2.1 Structure of Bt Crystal toxins 
Bt Cry proteins (?-endotoxins) consist of 3 domains (Li et al. 1991, Bravo 1997, 
Crickmore et al. 1998, de Maagd et al. 2003). Domain I is a seven ??helix bundle in 
which the central helix is completely surrounded by six outer helices (Schnepf et al. 
1998). This domain is the most conserved and is important for channel formation in the 
membrane. Domain II consists of three antiparallel ? -sheets sharing similar topology in a 
Greek key conformation, forming a ?-prism (Rajamohan et al. 1996). This domain is the 
most divergent, plays an important role in receptor interaction (Cheng and Nickerson 
1996, Jenkins and Dean 2000) and is the putative specificity-determining domain (Liang 
and Dean 1994). Finally, the third domain is a ? ?sandwich of two antiparallel ? ?sheets 
and is believed to be involved in toxin stability (Li et al. 1991, de Maagd et al. 1999b). 
 
1.3.2.2 Bt toxin mode of action  
Bt Cry proteins are stomach poisons primarily affecting mature columnar midgut 
epithelial cells after ingestion (Gill et al. 1992).  A generally accepted model for Cry 
toxin action is that of a multistage process (Himeno et al. 1985, Haider and Ellar 1989). 
Within the crystal, insecticidal proteins interact through hydrogen bonding, disulfide 
linkages, and hydrophobic interactions (Choma and Kaplan 1990, 1992). In lepidopteran 
insects, insecticidal proteins are released in the alkaline gut and hydrolyzed to toxins by 
proteases (H?fte and Whiteley 1989). An unusual feature is that activation of the protoxin 
appears to occur by a sequential series of proteolytic cleavages, initiated at both termini 
and proceeding towards the center of the protein until finally the protease-stable toxin is 
generated (Choma et al. 1990). Twenty five -30 amino acids from the amino and 
10 
approximately half of the remaining protein from the carboxyl-terminal are 
proteolytically cleaved (Bravo et al. 2002).  
 
Proteases such as trypsin (Pang et al. 1999), chymotrypsin and elastase are 
associated with the hydrolysis of the protoxins (Oppert 1999).  Generally 130 -140 k Da 
protoxin is activated into 60-70 k Da proteins (Bravo 1997). Although proteolytic 
activation generates the proteinase-resistant activated toxin, exceptions have been 
reported in many insects. Intramolecular processing has been reported for several Cry 
toxins. Cry1Ab (Convents et al. 1991), Cry2Aa (Audtho et al. 1999), Cry4Aa (Yamagiwa 
et al. 1999), Cry4B (Zalunin et al. 1998) and Cry9Aa (Zalunin et al. 1998) are cleaved in 
domain I, while Cry11Aa (Dai and Gill, 1993), Cry1Ac (Choma et al. 1990) and Cry1Aa 
(Pang et al. 1999) are cleaved within domain II.  The extra processing resulted in the 
production of a protein with less or no toxicity, which could increase the potential for Bt 
resistance development (Miranda et al. 2001). However, the extra nick in Cry3A renders 
it more soluble under neutral pH conditions making it more toxic to insects (Carroll et al. 
1997). 
 
Activated toxin binds to receptors present in brush border membranes (Hofmann 
et al. 1988, van Rie et al. 1990a). Later it undergoes conformational changes leading to 
oligomerization and enters the cell facilitated by pores formed in the membrane (Knowles 
1994). This allows the movement of K
+
 ions in to the gut lumen leading to an increase in 
hemolymph K
+
 concentration resulting in gut pH changes. Ultimately, the affected cells 
are destroyed due to high gut pH and osmotic lysis resulting in insect death (Li et al. 
1991, Lorence et al. 1995, Bravo 1997, Whalon and Wingerd 2003). 
11 
 
1.3.3 Insect midgut and response to Bt toxins 
The midgut epithelium of larval lepidoptera consists of a highly folded pseudostratified 
epithelium separated from a framework of muscle and trachea by a thin basement 
membrane (Chapman 1998). Within the epithelium are three main cell types: cylindrical 
columnar cells exhibiting apical (lumenal) microvilli; pear shaped goblet cells; each with 
a central cavity that opens to the gut lumen through a valve, and small round stem cells 
that are located between the bases of the columnar and goblet cells. Stem cells undergo 
mitotic activity immediately prior to each molt (Baldwin and Hakim 1991). Until the 
prepupal molt, stem cells differentiate into gut epithelial cells for the next instar. At the 
prepupal and adult molts the stem cells differentiate to phenotypes that characterize pupal 
and adult midguts (Engelhard et al. 1991, Loeb and Hakim 1996, Loeb et al. 2001, 2003). 
 
Histopathological studies have indicated dose-dependent destruction of cultured 
midgut cells from H. virescens larvae in the presence of Bt toxins (Loeb et al. 2001). 
After 2 days of exposure to 0.8 pg/ ?l AA 1-9 or 0.06 pg/ ?l HD-73, columnar and goblet 
cell numbers declined to ca 20% of controls. In contrast, stem cell numbers increased 
140-200% greater than the controls. The dynamics of depletion and replacement 
depended on toxin type and concentration. Two days after toxin removal, cell type ratios 
returned to approximate pre-toxin levels. The response of cultured midgut cells to Bt 
toxin injury was similar to injured vertebrate tissues dependent on stem cells for 
replacement and healing (Loeb et al. 2001). Similar observations were also made in 
dipteran insects when they were challenged with Bt israeliensis (Bti) proteins. 
Investigations using Simulium vittatum larvae infected with Btk HD 255 (Lacey and 
12 
Federici 1979) and S. variegatum infected with Bti (Rey et al. 1998) demonstrated 
morphological lesions in the intestinal epithelium which exhibited swollen cells, 
degenerated brush borders, disorganized nuclei, enlargement of intercellular spaces and 
cell lysis. Light and electron microscope observations in Simulium pertinax, a common 
black fly, revealed by time and endotoxin concentration, increasing damage of the larva 
midgut epithelium. The most characteristic effects were midgut columnar cell 
vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen 
and finally, cell death (Smouse and Nishiura 1997, Cavados et al. 2004).  
 
1.3.4 Transgenic crops  
The use of Bt to control insect pests is not new. Commercial insecticides containing Bt 
and its toxins (e.g., Dipel
?
, Thuricide
?
, Vectobac
?
) have been in the market for over 40 
years. Bt-based insecticides are considered safe for mammals and birds, and almost all 
non-target insects (and typically safer than most conventional insecticides). What is 
relatively new is that Bt crops contain a modified version of the Cry gene that has been 
incorporated into the plant's DNA, so that the plant's cellular machinery produces the 
toxin (Li et al. 2003). When a susceptible insect feeds on a leaf, bores into a stem, or 
feeds on essentially any other tissue of a Bt-containing plant, it ingests toxin and will 
either severely stunt or die within a few days. 
 
Numerous crop plants have been transformed to express Bt Cry proteins under the 
influence of constitutive promoters such as CaMV35S (Perlak et al. 1990, 2001). 
Initially, Bt crops were not very effective due to relatively low expression of insecticidal 
proteins, however, a combination of methods including the use of plant stable Bt Cry 
13 
proteins, plant-specific promoters and different expression methods (e.g. expression in 
chloroplast DNA as compared to nuclear DNA) (Kota et al. 1999) have changed the 
scenario.  
 
The US Environmental Protection Agency (EPA) classifies Bt crop plants under 
the Plant Incorporated Protectants (PIP) category. PIPs are pesticidal substances 
produced by plants and the genetic material necessary for the plant to produce the 
substance. The current list of Bt crop plants registered for commercial cultivation can be 
found on the EPA?s webpage at 
http://www.epa.gov/pesticides/biopesticides/pips/pip_list.htm and the currently registered 
PIPs for experimental use can be found at 
http://www.epa.gov/pesticides/biopesticides/pips/current_pip_eups.htm  
 
1.3.4.1 Bt cotton and its commercialization 
The first generation of Bt cotton, Bollgard
?
 produces Cry1Ac that has been rapidly 
adopted by growers since its commercial introduction into the US in 1996. Bollgard
?
 
provides effective protection from feeding damage by lepidopteran insect pests such as 
H. virescens, P. gossypiella and H. zea (Gould 1998). Bollgard
?
 has been approved for 
commercial cultivation in nine countries (James 2005) including two developed countries 
such as USA and Australia and seven developing countries namely, Argentina, China, 
Colombia, India, Indonesia, Mexico and South Africa (Sivasupramaniam et al. 2007). 
First generation Bt cotton is referred to as Ingard
?
 in Australia whereas in all other 
countries it is called as Bollgard
?
 (James 2005). The global adoption of Bollgard
?
 has 
increased dramatically from 800,000 ha in its first year of introduction (1996) to 5.7 m ha 
14 
in 2003.  Bt cotton acreage has increased tremendously in the USA from 0.8 to 2.8 m ha 
in 2006 accounting for 52% of total cotton acreage (Brookes and Barfoot 2006).  Its 
adoption has helped US farmers manage cotton insect pests effectively and increase their 
net income by $50/Ha, thereby increasing the total net value of US cotton production by 
$103 million in 2001. In addition, it is also safer to the environment including a 841.5 
metric tonnes reduction in insecticide active ingredients per year (Perlak et al. 2001, 
James 2002, Chitkowski et al. 2003, Mendelsohn et al. 2003, Head et al. 2005).  
 
Bollgard
?
 II, Event 15985 was developed by inserting the Cry2Ab2 gene to 
Bollgard
?
 (DP50B) (Greenplate et al. 2003). Bollgard
?
 II was introduced into Australia 
in 2002 and the USA in 2003.  This dual gene cultivar has expanded the range of benefit 
to growers and the environment. Bollgard II
?
 provides equivalent or increased control of 
major target pests of cotton compared to Bollgard
?
, with additional control of secondary 
lepidopteran insect pests such as beet armyworm Spodoptera exigua (H?bner) and fall 
armyworm Spodopeta frugiperda (Smith) (Sivasupramaniam et al. 2008). Pyramided Bt 
crops also provides an added dimension to effective resistance management (Roush 1998, 
Zhao et al. 2003).  Bt cotton varieties expressing Cry1F and Cry1Ac have been 
commercialized and marketed by Dow AgroSciences, and additional varities expressing 
VIP3A and Cry1Ab are being developed by Syngenta, respectively and should be 
available commercially within the next 1-3 years. VIP3A is a vegetative insecticidal 
protein produced by B. thuringiensis during vegetative growth, and represents a new 
family of insecticidal proteins (Lee et al. 2003).  One of the interesting features of Vip3A 
is that it shares no sequence homology with known ?-endotoxins (Estruch et al. 1996). 
15 
The mode of action of Vip3A has been examined and has been shown to target the 
midgut epithelium, where binding to midgut cells is followed by progressive 
degeneration of the epithelial layer (Yu et al. 1997). Receptors binding to VIP3A do not 
bind Cry proteins ensuring values in future resistance management plans.  
 
Both Cry2Ab2 and Cry1F found in Bollgard
?
 II and Wide Strike, respectively, are 
less toxic (susceptibility values are in the next paragraph) to H. zea compared to Cry1Ac 
(Sivasupramaniam et al. 2008). However, the low toxicity has been overcome by 
expressing toxin at higher levels (Greenplate et al. 2003). 
  
1.3.4.2 Insecticidal protein expression 
Bollgard
?
 expresses Cry1Ac in all above ground tissues and throughout the season. 
Expression levels vary in different plant tissues (Greenplate 1999, Greenplate et al. 2003, 
Kranthi et al. 2005, Sivasupramaniam et al. 2008), crop growth stages (Greenplate et al. 
2003, Bird and Akhurst 2005) and varieties (Adamczyk and Sumerford 2001, Adamczyk 
et al. 2001) and/or hybrids (Kranthi et al. 2005). Bollgard
?
 leaves have the highest 
expression of toxin concentrations followed by squares, bolls and flower parts 
(Greenplate 1999, Kranthi et al. 2005). 
  
1.3.4.3 Susceptibility of H. zea to different Bt proteins found in Bt cotton 
H. zea has a naturally higher tolerance to Cry1Ac than the other two target species, P. 
gossypiella and H. virescens, with wide variation in susceptibility among field 
populations (10-40-fold) (Stone and Sims 1993, Luttrell et al. 1999, Ali et al. 2006).  H. 
armigera (Liao et al. 2002, Akhurst et al. 2003) is known to have similar susceptibility 
(LC
50
=10?g/g) to Cry1Ac as H. zea (MacIntosh et al. 1990). Wide variation (71-fold) 
16 
(LC
50
: 0.01 to 0.71 ?g/ml) in susceptibility of H. armigera has been observed in India 
(Kranti et al. 2001, Jalali et al. 2004, Krishnappa et al. 2005).   
 
Cry2A (LC
50
=375.78ng/larvae) is less toxic to H. zea compared to Cry1Ac 
(LC
50
=63.60ng/larvae) (Karim et al. 2000). Similar observations were also reported by 
Sivasupramaniam et al. (2008) reported that Cry2Ab2 (LC
50
=17.476 ?g/ml) is less toxic 
to H. zea than Cry1Ac (LC
50
=0.87 ?g/ml).Among the Cry2A toxins, Cry2Aa is less toxic 
(LC
50
=681 ng/diet cup) compared to Cry2Ab2 (LC
50
=364 ng/diet cup) (Dankocsik et al. 
1990), which is present in the Bollgard
?
 II.  
 
Cry1F is another crystal protein found in new varieties of Bt cotton from Dow 
Agrosciences. Cry1F has relatively low toxicity (LC
50
>57.0 ng/mm
2
 of diet surface) to H. 
zea however it is highly effective against O. nubilalis (LC
50
=0.27) and S. exigua 
(LC
50
=25.6) (Chambers et al. 1991). 
 
VIP3A is vegetative insecticidal protein in VIPCOT
?
, a new Bt cotton event 
being developed by Syngenta has an LC
50 
between 112.5 and
 
420 ng/ cm
2
 against H. zea 
(Estruch et al. 1996, Lee et al. 2003).  
 
1.3.5 Selection for Bt resistance 
Insect strains resistant to Bt proteins have been selected from laboratory or field-collected 
insects (Table 1). Different selecting agents such as laboratory-produced or commercially 
available Bt proteins were used as selection agents. For laboratory produced Bt proteins: 
In some instances, genetically transformed bacteria such as E. coli, Pseudomonas 
fluorescens, and Bacillus etc. were used for generating Bt protein inclusion bodies.  In 
17 
some cases these inclusion bodies were soluble at alkaline pH (10.5) releasing protoxins 
which were, sometimes, activated using either trypsin or chymotrypsin to form activated 
toxin. All of these different forms such as inclusion bodies, protoxin and toxins have been 
used in various insect selection experiments. 
 
Commercial Bt formulations such as MVP II (containing Cry1Ac protoxin 
inclusion bodies encapsulated in Pseudomonas fluorescens cells), and Dipel
?
 (containing 
the HD-1 strain of Btk) Xentari
?
 (containing Bt aizawai) have been used as selection 
agents for developing resistant insects.  As will be discussed later, the use of formulations 
may add at least another variable into resistance selection due to the various non-
described ingredients found in formulations.  
 
  First instars were selected either individually or en masse either by incorporating 
Bt proteins into the diet (diet incorporation), overlaying Bt proteins onto the diet surface 
(diet overlay) or using different tissues from Bt-crops. Bioassay exposure times (7-21 D) 
varied across insect species depending on their biology and susceptibility to Cry toxins. 
Different selection criteria such as survival and growth inhibition were followed, and 
selected insects were reared on diet containing no Bt-protein until pupation. Resistance 
was determined by conducting bioassays and comparing these results with bioassays 
conducted against a parent susceptible population and thereby developing resistance 
ratios (RR). 
18 
1.3.6 Frequency of resistant alleles 
The risk of rapid pest adaptation to an insecticide is highly dependent on the initial 
frequency of resistance alleles in field populations and strategies for delaying pest 
resistance are based primarily on theoretical models using these frequencies. One key 
assumption of such models is that genes conferring resistance are rare (less than 10
-6
) in 
the field. The frequency of resistance alleles has been measured through field sampling of 
P. gossypiella larvae, genetic crossing of field collected males with laboratory selected 
Cry1Ac-resitant females in H. virescens, and female H. zea moths collected from light 
traps in the field. Theoretical models using this data predict that these insects can develop 
resistance to Cry1Ac very quickly in a span of three-four years (Storer et al. 2003). 
However, Bt resistance has not been observed even after a decade of Bt cotton 
cultivation.  
 
The estimated resistance allele frequency in H. virescens through individual 
mating of over 2,000 male moths collected in four states to females of a Bt toxin-resistant 
laboratory strain, was 1.5 X 10
-3
(Gould et al. 1997). The estimated frequency of a 
recessive allele conferring resistance to Cry1Ac was 0.16 (95% confidence interval [CI] 
= 0.05-0.26) in strains of PBW derived from 10 Arizona cotton fields during 1997. 
However, this frequency of resistance allele in P. gossypiella, has not increased since 
1997 even after higher adoption of Bt cotton in Arizona, and the frequency has remained 
low; between 0.05 and 0.11 during 1998 and 1999, respectively (Tabashnik et al. 2000). 
Although some variation occurred from 1999 to 2003,
 
the mean resistance allele 
frequency has not differed significantly
 
between 1998 and 2004 (0.004, 95% CI = 0-0.01) 
(Tabashnik et al. 2005b). The estimated non-recessive allele frequency of H. zea during 
19 
2000 was 4.3 X 10
-4
 and 3.9 X 10
-4
 to Cry1Ac and Cry2Aa, respectively (Burd et al. 
2003). Initial frequencies of alleles conferring resistance to transgenic Bt poplars 
producing Cry3A in a natural population of the poplar pest Chrysomela tremulae F. was 
estimated to be 0.0037 (average over three years) for the period 1999-2001 (95% CI = 
0.00045-0.0080) (Genissel et al. 2003).  To date, extensive screening of European corn 
borer, O. nulialis the major pest targeted by first generation of Bt corn, has not identified 
any individuals with alleles conferring resistance to Bt corn (Andow et al. 1998, 2000, 
Bourguet et al. 2003). 
  
1.3.7 Resistance mechanisms 
The mode of action of Bt toxins suggests at least three possible physiological or 
biochemical mechanism(s) of resistance (Ferre et al. 1995, Ferre and van Rie 2002, 
Griffits and Aroian 2005). The first involves pH- and protease-mediated dissolution and 
activation of the crystal. Enzymatic changes in the resistant insect gut may have resulted 
in the detoxification of Bt proteins, or the inability to activate them. Secondly, changes 
may have occurred in the gut cell membrane, interfering with binding of the toxic moiety. 
Thirdly, cellular changes may have occurred that influence the sensitivity of the cell to 
pore formation or their capacity to recover from toxin effects.  
 
1.3.7.1 Altered proteolytic processing 
Serine proteases, such as trypsin, chymotrypsin and elastase are important in both 
solubilization and activation of Bt protoxins (Oppert 1999). In some insects the altered 
activities of these proteases have resulted in resistance development.  Gut protease 
activity has been altered in Bt subspecies entomocidus (Bte HD-198) resistant strain 198
r
 
20 
of Indian meal moth Plodia interpunctella (H?bner) (Oppert et al. 1996).  Western blot 
analysis showed that 198
r
 enzymes were much less effective than Bt susceptible (688
s
) or 
Bt subsp kurstaki (strain HD-1, Dipel
?
) resistant strain (Dpl
r
) in hydrolyzing protoxin. In 
addition, protoxin hydrolysis produced many non-toxic intermediates with a different 
proteolytic pattern, and proteolysis was incomplete even after 4 h incubation as compared 
to complete hydrolysis in the other two strains.  The proteolytic enzymes were shown to 
be trypsin-like enzymes (Oppert et al. 1997). Assays conducted using the trypsin 
diagnostic substrate, BApNA (N-?-benzoyl?L-arginine p-nitroanilide), revealed reduced 
activities of enzymes from Bt subsp aizawai (133
r
) and entomocidus (198
r
) resistant 
strains. The specific activity of gut proteases from these strains was less than one-half of 
those in the kurstaki-resistant and susceptible strains.  Activity from the kurstaki resistant 
strain was approximately 30% higher than the activity from the parent susceptible strain, 
suggesting adoption of different resistant mechanisms by this strain. 
 
Two (~45 and ~25 k Da proteins) major BApNA hydrolyzing enzymes were 
identified from gut extracts of susceptible insects, and absent in both resistant strains. A 
subsequent study demonstrated a genetic linkage between decreased susceptibility to 
Cry1Ac and the absence of a major gut protease. Moreover, the involvement of changes 
in midgut proteases in resistance was further affirmed by the observation of 11-fold 
higher resistance levels for Cry1Ab protoxin than for Cry1Ab toxin in the 198
r
 strain 
(Herrero et al. 2001). 
 
Reduced trypsin-like proteinase activity has been also studied in the activation of 
protoxins by four selected (KS-SC, KS-NE, IA-1, and IA-3) and one susceptible (IA-S) 
21 
strains of European corn borer O. nubilalis. The hydrolyzing efficiencies were compared 
using three synthetic substrates, BApNA for trypsin-like, N-succinyl-ala-ala-pro-phe p-
nitroanilide (SAAPFpNA) for chymotrypsin-like and N-succinyl-ala-ala-pro-leu p-
nitroanilide (SAAPLpNA) for elastase-like proteinase activities (Huang et al. 1999a).  
 
Enzyme kinetic studies of trypsin-like proteases revealed no change in Michealis 
constants (K
m
) among five strains but V
max
 decreased by 35% in the KS-SC resistant 
strain as compared to the IA-S.  However, the detectable reduction was observed in the 
hydrolysis of protoxin in the KS-SC strain compared with the IA-S strain.  At the same 
time, no significant differences were found in trypsin activity between the IA-S strain and 
the three other resistant strains (i.e., KS-NE, IA-1, and IA-3). Similarly there were no 
detectable differences in the activity of chymotrypsin among all strains examined 
suggesting different resistance mechanisms in the other three strains (Huang et al. 1999).  
 
In a subsequent study, two forms (soluble and membrane fractions) of trypsin-like 
proteases were identified (Li et al. 2004a).  Serine proteases from soluble fractions of the 
susceptible strain were more active than those of the resistant strain (KS-SC).  When 
casein was used as a substrate for analysis no significant differences were observed 
between different fractions of proteases. However when Cry1Ab protoxin was used as a 
substrate, zymogram analysis indicated that approximately 20% less protoxin was 
hydrolyzed with soluble extracts from the resistant strain when compared with similar 
extracts from the susceptible strain.    
Enzymes from the CP73-3 strain of H. virescens resistant to Bt subsp kurstaki 
(HD-73) were reported to process more slowly, and to degrade toxin faster than enzymes 
22 
from the susceptible strain (Forcada et al. 1996).  In the NO-95C colony of P. xylostella, 
resistant levels for crystalline Cry1Ca protoxin were about 2.5-fold higher than for 
Cry1Ca toxin. Thus, reduced conversion to toxin is a minor mechanism of resistance in 
this strain (Liu et al. 2000).  
 
1.3.7.2 Receptor binding 
Four different receptors have been identified as binding sites of Bt Cry toxins in insects. 
Among the four, cadherin-like proteins with molecular masses between 175 & 220 k Da 
(Vadlamudi et al. 1993, 1995; Nagamatsu et al. 1998a, 1998b; Dorsch et al. 2002, 
Flannagan et al. 2005) and  aminopeptidase N (APN) with molecular masses in the range 
of 108 to 252 k Da (Sangadala et al. 1994, Knight et al. 1995, Cheng and Nickerson 
1996, Luo et al. 1996, 1997; Cooper et al. 1998, Yaoi et al. 1999, Banks et al. 2001, 
Agrawal et al. 2002, Hossain et al. 2004, Liu et al. 2004) are the most studied receptors. 
These receptors are known to preferentially partition into lipid rafts (Zhuang et al. 2002). 
Recently anionic glyconjugate (Valaitis et al. 2001), actin & alkaline phosphatases 
(McNall and Adang 2003, Jurat-Fuentes and Adang 2004), 252 kDa (P252) protein 
(Hossain et al. 2004), and glycolipids (Griffits et al. 2005, W. Moar, Auburn University, 
personal communication) are added to the possible candidates for binding sites. The 
binding site modification results either through a reduction in the quantity of binding sites 
or through an alteration in the receptor itself, affecting the sensitivity for binding (Herrero 
et al. 2005).  
 
The reduction in binding affinity was first reported for a Dipel
?
-resistant P. 
interpunctella strain (343
r
) (van Rie et al. 1990).  Binding studies with brush border 
23 
membrane vesicles (BBMV) revealed a 50% reduction in binding affinity (K
d 
= 36.3 ? 
22.7nM) for Cry1Ab in 343
r
, however no differences were observed with respect to the 
number of binding sites (R
t
=1.77 ? 0.58 pmol/mg of membrane protein) compared to the 
susceptible strain. Binding affinity was similar in both strains when Cry1Ca was used but 
the R
t
 value (0.38 ? 0.07 and 1.15 ? 0.20 pmol/mg for susceptible and resistant strains, 
respectively) was significantly higher (three-fold) in the resistant strain. This shows that 
the observed resistance was primarily due to an alteration in the binding site for Cry1Ab 
and concurrently, the increased affinity to bind Cry1Ca explains its susceptibility to this 
toxin. 
 
Differences in binding affinity were also observed in resistant (Dpl
r
 and 198
r
) and 
susceptible (688
s
) strains of P. interpucntella to Cry1Ab (Herrero et al. 2001). The Dpl
r
 
(K
d 
=14.48nM) strain showed a 60-fold reduction in binding affinity compared to 688
s
 
(K
d 
=0.25nM). Conversely, no significant differences were observed between Dpl
r
 and 
198
r
 binding of 
125
I-Cry1Ac. These results suggest that the same change in the Cry1A 
binding site had different effects on Cry1Ab and Cry1Ac binding. Whereas Cry1Ab 
affinity would be reduced, the change could only affect post binding steps of the Cry1Ac 
mode of action, such as membrane insertion or pore formation. In contrast to strains 343
r
 
and Dpl
r
, strain 198
r
 showed only a slight reduction in binding of Cry1Ab (five-fold 
higher K
d
 and three-fold lower R
t
). 
 
Competitive binding analysis revealed two binding sites for Cry1A toxins in three 
resistant (NO-QA, PEN & PHI) and one susceptible (LAB-V) strain of P. xylostella. The 
reduction in binding sites for Cry1Ab and Cry1Ac was reported as a cause for resistance 
24 
in NO-QA and PEN strains; whereas binding of Cry1Aa was unaltered in all three strains 
(Tabashnik et al. 1997).  Further analysis of Cry1Aa binding with PHI indicated a good 
fit for the two binding sites model (Ballester et al. 1999). The two K
d
 values (K
d1
=0.3 ? 
0.1 & K
d2
= 20.3 ? 4.4 nM) for binding to two sites were essentially the same as the K
d
 
values obtained for the susceptible strain (K
d1
= 0.1 ? 0.1 & K
d2
= 17.7 ? 1.0 nM). On the 
other hand, homologous binding studies with NO-QA and PEN strains fit the two binding 
sites model. The K
d
 and R
t
 values of Cry1Aa for NO-QA (4.8 ? 2.7 nM and 1.9 ? 0.8 
pmol/mg of protein, respectively) and PEN (4.0 ? 2.8 nM and 1.9 ? 0.8 pmol/mg of 
protein, respectively) were similar to each other and were intermediate between the 
values obtained for the two binding sites of LAB-V and PHI. 
A four-fold decrease in binding affinity for Cry1Ac was observed in AZP-R 
(Cry1Ac-resistant PBW) compared to the APHIS-S (susceptible) strain.  Though the 
concentration of binding sites (R
t
) was 23-fold higher in the resistant compared to the 
susceptible strain, the ratio (R
t
/K
d
) of binding site concentration to dissociation constant 
did not differ significantly among the two strains (Gonzalez-Cabrera et al. 2003) 
suggesting that the reduction in the binding affinity is the primary mechanism of insect 
resistance to Cry1Ac in PBW. 
In H. armigera Cry1Aa, Cry1Ab and Cry1Ac share common binding sites. 
Binding experiments in the presence of concanavalin A showed that Cry1Ac and Cry1Ab 
bind to different epitopes (Estela et al. 2004).  
 
Reduction in binding site numbers was observed in Cry3Aa-resistant Colorado 
potato beetle Leptinotarsa decemlineata. Saturation binding studies with BBMV and 
125
I-
25 
Cry3Aa revealed approximately 60% less binding of Cry3Aa toxin in the resistant strain 
compared to the susceptible strain. Nonetheless competitive binding assays showed no 
differences, suggesting observed resistance was due to changes in the number of binding 
sites (Loseva et al. 2002).   
 
1.3.7.2.1 Causes for reduction in binding in resistant strains 
A mutation that resulted in the deletion of eight amino acids in three alleles (r1, r2 & r3)  
of the cadherin-like protein was shown to be associated with Cry1Ac resistance in P. 
gossypiella (Morin et al. 2003). However, a mutation that resulted in the deletion of only 
one amino acid resulted in reduced binding on the cadherin-like receptor in Cry1Ac-
resistant H. virescens (Xie et al. 2005). The mutated amino acid sequence described 
above overlaps the Cry1Ab binding site (1363 to 1464) in tobacco hornworm, Manduca 
sexta (L.).  The single amino acid mutation in mutant line L1425R from CTG to CGG 
was responsible for the observed reduction in binding.   
 
1.3.7.2.2 Identification of resistance genes 
The identification and isolation of Bt resistant genes has been met with little success.  
Genetic mapping experiments with the laboratory-selected YHD-2 resistant strain of H. 
virescens showed a tight linkage between resistance to Cry1Ac and a cadherin encoding 
gene Bt-R4 or HevCaLP.  Insertion of a retrotransposon disrupting Bt-R4 in the YHD-2 
strain leads to a high level of resistance >10,000 fold, linking this protein to Bt resistance 
(Gahan et al. 2001).  
26 
1.3.7.3 Other resistance mechanisms 
Ingestion of a sublethal dose of Cry1Ac by fourth instar CP73-3 H. virescens larvae 
resulted in similar histopathological changes in columnar gut cells compared with cell 
damage in susceptible larvae (Martinez-Ramirez et al. 1999). Likewise, larvae from both 
a susceptible colony and another resistant H. virescens colony (KCB) showed comparable 
midgut epithelium damage following Cry1Ac ingestion (Forcada et al. 1999). Therefore, 
it is possible that resistance in CP73-3 and KCB is due to a more efficient repair 
(replacement) of damaged midgut cells. 
 
1.3.8 Inheritance of resistance 
Resistance development can be delayed by decreasing the dominance of resistance, 
provided resistance is inherited as a recessive character (Tabashnik et al. 2003, 2004a, 
2004b; 2005b). Inheritance can be determined by four different methods; testing on Bt 
crops, use of leaf-dip, diet incorporated or overlay bioassays (Tabashnik 1991). Among 
these, bioassays using Bt crops are the most realistic of what happens in the field. Leaf-
dip bioassays simulate the situations rather well in the field because insects ingest Bt 
proteins or spore/crystal mixtures along with the fresh plant material. Finally, bioassays 
(diet incorporation or diet overlay) using artificial diet are the least similar to the field 
situation. 
 
The degree of dominance (D), dominance (D
LC
) and effective dominance (D
ML
) 
of resistance have been calculated by following Stone (1968) , Bourguet et al. (2000) or 
many other methods (Preisler et al. 1990).  All of these values are based on LC
50?
s in 
which D = (2X
2
 - X
1
- X
3
) /(X
1
 - X
3
), where X
1
, X
2
, and X
3
 are the logarithms of the 
LC
50?
s for the resistant homozygotes, heterozygotes, and susceptible homozygotes, 
27 
respectively. D values range from -1 (completely recessive resistance) to 1 (completely 
dominant resistance). D
LC
=(D+1)/2 and D
ML
=(ML
RS
-ML
SS
)/(ML
RR
-ML
SS
). D
LC
 is the 
estimate of dominance with 0 for completely recessive, 0.5 for semi-dominant and 1.0 for 
completely dominant trait. D
ML
 defines the effective dominance of survival where ML
RR
, 
ML
SS
, ML
RS
 are the % mortality levels of the resistant, susceptible and hybrid progeny on 
Bt crops.  
 
Resistance to Cry1Ac was reported to be autosomal and inherited as incompletely 
recessive in H. armigera (D
LC
=0.26) (Akhurst et al. 2003), P. xylostella (Tabashnik et 
al.1997, Tang et al. 1997), H. virescens (Gould et al. 1992, 1995), P. gossypiella (D=-
0.61) (Tabashnik et al. 2002a) and Trichoplusia ni (H?bner) (D=-0.402) (Janmaat et al. 
2004, Kain et al. 2004).  However, recent studies from Australia (Bird and Akhurst 2005) 
and India (Kranthi et al. 2006) suggest that Cry1Ac resistance is inherited as a semi-
dominant trait in H. armigera. A similar observation has also been made with H. zea 
(Burd et al. 2003, W. Moar, Auburn University, personnel communication). Cry1C 
resistance was inherited as a recessive character in a P. xylostella (D= 0.26) strain that 
developed high levels of resistance to Bt subsp. kurstaki in the field (Liu and Tabashnik 
1997b, Zhao et al. 2000) (D=-0.22) and the realized heritability of resistance was 0.10. 
However, in some insects Bt resistance is inherited as semi-dominant or dominant 
character (Tabashnik et al. 2000). Incomplete dominance was observed in O. nubilalis to 
Dipel (Huang et al. 1999b), and P. xylostella to Cry1Ac (Sayyed et al. 2000). Dominance 
increased as the concentration of Bt proteins decreased, suggesting the possibility of a 
28 
single resistance gene controlling resistance with 3 or more alleles, which might hasten 
the rate of resistance development.  
 
1.3.9 Fitness costs 
The development of resistance in any insect is often associated with decreased fitness. 
The fitness of a resistant strain may be affected in terms of incubation period, larval 
duration, pupal duration, adult longevity, fecundity, fertility hatchability, diapause 
survivability etc.  
 
Reduced survival (51.5%) of Cry1Ac-resistant PBW was observed on non Bt 
(NBt) cotton compared to susceptible strains; however, there was no difference in their 
developmental time. Crosses between resistant and susceptible strains indicated that 
survival costs could be dominant (Carriere et al. 2001b). Additionally, the emergence of 
PBW moths from overwintering pupae was greatly (71% reduction) affected in the 
Cry1Ac-resistant strain compared to the susceptible strain (Carriere et al. 2001a). 
 
Resistant larvae feeding on Bt cotton required an average of 5.7 D longer to 
develop than susceptible larvae on NBt cotton. This developmental asynchrony, 
therefore, favors assortative mating among resistant moths from Bt plants. In the field, the 
extent of developmental asynchrony and assortative mating would be affected by 
variation in toxin expression in plant tissues and crop phenology, weather and the 
overlapping of generations (Liu et al. 1999). 
 
Reduced developmental time and pupal weight was observed in Cry1Ac-resistant 
P. gossypiella when they were tested on increasing concentrations of Cry1Ac in artificial 
29 
diet.  In addition, tests conducted on NBt cotton using resistant and susceptible strains 
indicated lower survivability, slower development, lower pupal weight and fecundity in 
the resistant strain compared to the susceptible strain (Liu et al. 2001c). 
 
Glasshouse experiments using Bt cotton showed that 50 and 62% of BX (Cry1Ac-
resistant) strain of H. armigera could complete larval development on initial (<15 weeks) 
and later (>15 weeks) crop stages, respectively. However, no susceptible larvae survived 
to pupation on Bt cotton plants.  However, their developmental rate was faster on NBt 
cotton compared to the BX strain, which took 7 D more. Though studies on NBt cotton 
indicated no developmental delay in F1 progeny of reciprocal crosses indicating recessive 
inheritance of resistance, results from studies on Bt cotton showed that F1 progeny can 
survive to later instars indicating partially dominant inheritance (Bird and Akhurst 2004). 
 
Fitness costs can vary in resistance strains on different host plants. Dipel
?
-
resistant T. ni performed differently on cucumber, tomato and pepper, among which 
pepper is least preferred host. None of the P
R
 (Dipel
?
 resistant strain) larvae survived on 
pepper, contrasting to more than 50% survivors on two other crops (Jaanmat and Myers 
2005).  
 
1.3.10 Cross resistance between Cry proteins  
Cross resistance is defined as tolerance to a usually toxic substance as a result of 
exposure to a similarly acting substance. The risk of cross resistance occurring between 
Cry proteins is substantiated because their mode of action is similar. Though, different 
Cry proteins can bind to different receptors, there are certain overlapping binding sites 
which can bind to more than one Cry protein. In one case, one gene was demonstrated to 
30 
confer resistance to four different Cry proteins (Tabashnik et al. 1997). Among those four 
Cry toxins, three (Cry1Aa, Cry1Ab, and Cry1Ac) were closely related and the other 
(Cry1F) displayed low sequence similarity with others. Available information on cross 
resistance is tabulated in Table 1.  
 
Cross-resistance patterns vary considerably among different insects and different 
Cry toxins. Selection for Cry1Ac resistance in H. virescens (YHD2) (Gould et al. 1995), 
and P. gossypiella (AZP-R and APHIS-98R) (Tabashnik et al. 2000, 2002) resulted in 
high levels of cross resistance to Cry1Aa and Cry1Ab and no or little cross resistance to 
Cry1Ca or Cry2Aa. Different strains of the same insect species have different cross 
resistance patterns though they were selected using the same Cry protein. Such examples 
can be observed in Cry1Ac resistant H. virescens strains; CP73-3 >50-fold resistant 
strain, showed 53-fold cross resistance to Cry2Aa (Gould et al. 1992), whereas YHD2 
strain with over 10,000-fold resistance to Cry1Ac did not have any cross resistance to 
Cry2Aa (Gould et al. 1995).  Similar cross resistance patterns were observed in P. 
gossypiella, AZP-R with 3100-fold resistance to Cry1Ac had no cross resistance to 
Cry1Ja (Tabashnik et al. 2000, 2002) on the other hand, APHIS-98R with just over 100-
fold resistance to Cry1Ac had little cross resistance to Cry1Ja (Tabashnik et al. 2000, 
2003). These two above-mentioned examples suggest that, probably in the initial stages 
of resistance development one might tend to see little or no cross resistance as the 
resistant population is not homogeneous compared to highly resistant strains. This 
observation has tremendous implications in the field as we see increased adoption of Bt 
crops expressing more than one Bt insecticidal protein. 
31 
 
P. xylostella strain NO-QA, which was selected for resistance to a mixed 
formulation of Cry1A toxins, also exhibited no cross resistance to Cry1Ca or Cry2Aa 
(Tabashnik et al. 1996, 1997). This condition of high level (>500-fold) resistance to 
Cry1A toxins that does not lead to cross resistance to Cry1Ca is defined as ?mode 1? 
resistance (Tabashnik et al. 1998). A similar mode was observed in P. interpunctella 
strain 198r, which was selected for resistance to a Bt strain expressing Cry1A, Cry1C and 
Cry1D proteins (McGaughey and Johnson 1994). These observations suggest two basic 
types of resistance, one exhibiting high-level, narrow-spectrum resistance, and the other 
featuring moderate-level, broad-spectrum resistance.  
 
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68
Table 1. Selected insect species and strains that have developed resistance to Bt proteins
*
 
 
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
  ES
d
 Formulation
/Toxin 
RR
e
 
P. 
interpunctella 
Dipel FSC 343-R 13 Dipel  100  McGauhey 1985 
    Btk HD-337 >100  McGaughey and 
Johnson 1987   Bte HD-198 1.1 
  Bta HD-133 1.4  
  36 Dipel >250 
? processing
McGaughet and 
Beeman 1988, Johnson 
et al.1990 
    Cry1Ab (877) 
? binding 
van Rie et al. 1990b 
Cry1Ca 0.3  
P. 
interpunctella 
Bte HD-
198 
SC 198r 19 HD-198 21   McGaughey and 
Johnson 1992 
    HD-198 32  McGaughey and 
Johnson 1994 Cry1Aa 10 
    Cry1Ab 27  
Cry1Ac 150 
    Cry1Ba  9  
Cry1Ca 5 
    Cry2A 20  
Cry1Ac-PT 128 
? activation 
Oppert et al.1994 
    Cry1Ab-PT 264  Herrero et al. 2001 
  Cry1Ab 25 
? binding 
  Cry1Ac n.d 
? binding 
 
 
 
 
 
69
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation
/Toxin 
RR
e
 
P 
interpunctella 
Bta HD-
133 
SC 133r 23 HD-133 61.5  McGaughey and Johnson 
1992 
   HD-133 94  McGaughey and Johnson 
1994 
  HD-1 45  
   Cry1Aa 17  
Cry1Ab 226 
  Cry1Ac 789 
     Cry1Ba 44  
 
  
 
Cry1Ca 19  
     Cry2A 24  
P. 
interpunctella 
Dipel FSC Dipel
r
 24 Dipel 70  McGaughey and Johnson 
1992 
     Cry1Ac 2816  McGaughey and Johnson 
1994 
     Cry1Ab-PT 1049  Herrero et al. 2001 
     Cry1Ab   290 
? ?binding 
P. xylostella Btk FSC BL?  Field Dipel 1  Ferre et al. 1991 
     Cry1Ab   >200 
? binding 
     Cry1Ba  2   
  Cry1Ca  0.5 
P. xylostella Btk FSC   Dipel 0.4  Ballester et al. 1994 
  Cry1Aa 1.3 
  Cry1Ab 236 
 
   Cry1Ac 1 
 
  Cry1Ba 
 
 
 
 
70
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation
/Toxin 
RR
e
 
 Cry1Ab/ 
Cry1Ac-
1Ab 
T/CC PHI  Cry1Aa >1 
? binding 
Ballester et al. 1999,  
Tabashnik et al. 1997 
     Cry1Ab >1 
? binding 
Tabashnik et al. 1997 
Cry1Ac  >1 
? binding 
Ballester et al. 1999, 
Tabashnik et al. 1997 
     Cry1Ca (1)  Tabashnik et al. 1997 
  Cry1Fa (1) 
Cry1Ja (1) 
P. xylostella Btk FSC NO Field Dipel 26.5  Tabashnik et al.1990 
 Dipel FSC NO-Q 9 Dipel 820  Tabashnik et al.1991 
   NO-Q 15 Dipel low  Tabashnik et al.1994 
 Dipel FSC NO-
QA 
 Dipel  3300 
? processing
Liu and Tabashnik 1997, 
Tabashnik et al. 1992, 
1993 
     Cry1Ac >59 
? binding 
Tabashnik et al.1994 
Cry1Aa  >100 
? binding 
Ballester et al. 1999,  
Tabashnik et al. 1996, 
1997 
     Cry1Ab   >100 
? binding 
Tabashnik et al. 1996, 
1997    
 
 
Cry1Ac   >100 
? binding 
     Cry1Ba 3  Tabashnik et al. 1996 
  Cry1Ca  2 
     Cry1Da  3   
Cry1Fa >100 
  Cry1Ia  3 
   Cry1Ja >140  
 
 
71
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation
/Toxin 
RR
e
 
     Cry1Ac-
canola   
98/0  Suresh et al. 1998 
 Cry1Ac NO-
QAGE 
  Cry1Aa  >20000  Tabashnik et al. 2000 
   Cry1Ab  >10000 
 Cry1Ac >40000  
 Cry1Bb 2 
   Cry1Ca  1 
 Cry1Da  
 Cry1Fa >10000 
   Cry1Ja >2000 
 Cry2Aa 5  
 Cry9Ca 2 
P. xylostella Btk /Bta FSC NO-95 Field Cry1Ca  22  Liu and Tabashnik 1997 
     Dipel  134  Liu et al. 1996 
  Xentari 3 
 Cry1Ca CC NO-
95C 
6 Cry1Ca 62  Liu and Tabashnik 1997 
 Cry1Ca T NO-
95C 
5 Cry1Ca 19 
? binding 
Liu et al. 2000 
    5 Cry1Ca 
crystal 
48  
 
 
 
 
 
 
 
 
72
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanis
m
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
P. xylostella Btk FSC Loxa A Field Javelin 300   Tang et al. 1996 
     Dipel 22  
    Cry1Aa >200  
     Cry1Ab  >200 
? binding 
    Cry1Ac >200  
     Cry1Ba 2.5 
    Cry1Ca  3.4  
     Cry1Da 1 
    Cry9Ca  1  Lambert et al. 1996 
     Cry1Ac-
broccoli 
95/20  Tang et al. 1999 
P. xylostella Btk /Bta FSC Cry1C-Sel Field Cry1Ca 31  
 
Zhao et al. 2000 
 Cry1Ca PT Cry1C-Sel 6 Cry1Ca 120 
 
Cao et al. 1999 
 Cry1Ca PT/Cry1C
broccoli 
Cry1C-Sel  Cry1Ca  12400 
? binding 
Zhao et al. 2000 
P. xylostella Btk FSC  Field   
 
Tabashnik et al. 
1997  Cry1Ac/ 
Cry1Ca 
SC PEN 6 Cry1Aa  High 
? binding 
    6  Cry1Ab High 
? binding 
Cry1Ac High 
? binding 
    6  Cry1Ca No 
 
Cry1Fa High 
 
    6  Cry1Ja >1000 
 
P. xylostella Btk /Bta FSC SERD3 Field Dipel 330 
 
Wright et al. 1997 
     Florbac 160 
 
    Cry1Aa n.d. 
? binding 
 
 
 
73
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanis
m
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
     Cry1Ab  n.d. 
? binding 
 
    Cry1Ac n.d. 
? binding 
 Dipel FSC Btk-Sel 3 Dipel 600 
 
    3 Florbac 60 
 
 Florbac FSC Bta-Sel 3 Florbac 300 
 
    3 Dipel 80 
 
P. xylostella Btk FSC UNSEL-
MEL 
Field Cry1Ab 121  
 
Sayyed et al. 2000 
     Cry1Ac 300 
 
    Dipel 40 
 
     Xentari 13 
 
 Cry1Ac Toxin 1AcSEL-
MEL? 
5 Cry1Ac 10500 
? binding  
    5 Cry1Ab 264 
? binding 
Dipel 59 
 
    5  
 
Xentari  10 
 
 Cry1Ab toxin 1AbSEL-
MEL? 
5 Cry1Ab 500 
 
Sayyed et al. 2000 
    5 Cry1Ac 7000 
 
 Dipel 81 
 
    5 Xentari 16 
 
 Dipel FSC BtkSEL-
MEL? 
5  Dipel 112 
 
    5   Cry1Ac 10700 
 
 5 Cry1Ab 900 
 
 
 
74
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanis
m
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
    5 Xentari 8  
 
 
 Xentari FSC BtaSEL-
MEL? 
5  Xentari 30  
 
    5  Cry1Ac 7260 
 
 5  Cry1Ab 420 
 
    5   Dipel 40 
 
P. xylostella Btk FSC ROO Greenho
use 
Toarow CT 704 
 
Hama et al. 1992 
     Thuricide 160 
 
Dipel  23 
 
     Bacilex 4 
 
H. virescens Cry1Ac/ T/ YHD2  Cry1Aa 32* ? binding
 
Gould et al. 1995, 
Lee et al. 1995 
 Cry1Ac CC  Cry1Ab >2300 
? binding 
    19 Cry1Ac >10000 ? binding 
   Cry1Fa CellCap 3700 
 
Gould et al. 1995 
  Cry1Ca 
CellCap 
2.5 
 
   Cry2Aa 25  
 
 Cry1Ac CC YHD2100
0MVP 
 Cry1Aa >20 
? binding 
 
     Cry1Ab  2000 ? binding  
Cry1Ac 
CellCap 
23,0000 
? binding 
 
     Cry1Fa   130 ? binding  
   Cry2Aa 9.5 
 
Kota et al. 1999 
 
 
 
 
75
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
H. 
virescens 
Cry1Ab CC SEL  22 Cry1Ab Cell 
Cap 
71 
? binding,  
? processing 
MacIntosh et al. 
1991 
 Dipel FSC  22 Cry1Ac 16 
? binding, 
? processing 
    22 Dipel 57 
 
H. 
virescens 
Cry1Ac T CP73-3 17 Cry1Ab 13 
  ? binding 
? activation 
? degradation 
? cell repair 
Forcada et al. 
1996,  
Gould et al. 1992, 
Martinez-ramirez 
et al. 1999 
    17 Cry1Ac 50 
? binding 
? cell repair 
Gould et al. 1992, 
Martinez-ramirez 
et al. 1999 
    17 Cry2Aa 53 
 
Gould et al. 1992 
 Cry2Aa PT CxC1000IIA 24 Cry1Ac >100 
 
Kota et al. 1999 
    24 Cry2Aa >330 
 
 24 Cry2Aa-cotton 0/n.d. 
 
H. 
armigera 
Cry1Ac PT BX 24 Cry1Ac 111 ?B Akhurst et al. 
2003     
Cry1Ab 157  
 Cry2Aa 1 
 
     Cry2Ab 1.4 
 
 Dipel 5 
 
     Xentari  7 
 
 MVP  69 
 
     HD73 
spore/crystal 188 
 
 
 
76
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
 Cry1Ac Cotton leaves  42 HD73 Cry1Ac -PT 1683.8 
 
Meng et al. 2004 
    42 Dipel 15.7 
 
  43 21% MVP II WP 1779.8 
 
    43 21% MVP II liquid  1233.4 
 
  Cotton leaves RC 22 Btk 3.1 
 
Lu et al. 2004 
    22 Cry1Ac 11 
 
  Btk RB 22 Btk 5.2 
 
    22 Cry1Ac 4.9 
 
O. 
nubilalis 
Dipel FSC KS-SC-R 7 Dipel 73  
 
Huang et al. 1997 
Cry1Ac CC S-I  8 Cry1Ac CellCap 162 
 
Bolin et al. 1999 
O. 
nubilalis 
Cry1Ab PT Europe-R 69 Cry1Ab 9.8 
 
Siqueira et al. 
2004     Cry1Ac 
35.4  
 Cry1Ab  RSST-R 41 Cry1Ab 9.0 
 
     Cry1Ac 52.6 
 
 Cry1Ab  Nebraska-R 50 Cry1Ab 1.9 
 
     Cry1Ac 7.2 
 
 Cry1Ab  Iowa-R 61 Cry1Ab 1.2 
 
     Cry1Ac 5.4 
 
S. exigua Btk HD-1 SC  20 Btk HD-1 1 
 
Moar et al. 1995 
S. exigua Cry1Ca IB/T  25 Cry1Ca 850 
? binding 
? NS 
binding 
    22 Cry1Ab 93 
 
 34 Cry2Aa 73 
 
    34 Cry9Ca 12 
 
 
 
 
77
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
S. 
littoralis 
Cry1Ca SC  14 Cry1Ca spcry >500 
 
Muller-Cohn et al. 
1996    14  Cry1Da spcry 7 
 
   14 Cry1Ea spcry 34 
 
  14 Cry1Fa spcry 1 
 
    14  Cry1Ab protoxin 3 
 
   14 Bta 7.29 spcry 7 
 
P. 
gossypiell
a 
Cry1Ac CC APHIS-98R  Cry1Ac CellCap >100 
 
Liu et al. 2001 
   Cry1Aa protoxin high 
 
Tabashnik et al. 
2000  Cry1Ab protoxin high 
 
    Cry1Ac protoxin high 
 
     Cry1Bb protoxin weak  
 
   Cry1Ca protoxin no 
 
   Cry1Da protoxin no 
 
   Cry1Fa protoxin no 
 
   Cry1Ja protoxin weak 
 
   Cry2Aa protoxin no 
 
   Cry9Ca no 
 
P. 
gossypiell
a 
Cry1Ac CC AZP-R  Cry1Ac Cell Cap 300 
 
Tabashnik et al. 
2000 
   Cry1Ac-cotton 40/1.6 
 
 Cry1Aa protoxin Yes 
 
    Cry1Ab protoxin Yes 
 
  Cry1Ac protoxin Yes 
 
    Cry1Bb protoxin Weak 
 
     Cry1Ca protoxin No  
 
   Cry1Da protoxin No 
 
   Cry1Fa protoxin No 
 
 
 
78
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
     Cry1Ja protoxin No 
 
 
 Cry2Aa protoxin No 
 
T. ni Cry1Ab T  7   Cry1Ab 31 
 
Estada and Ferre 1994 
    Cry1Aa 1 
 
  Cry1Ac 0.5 
 
C. scripta Cry3Aa CC  35 Cry3Aa CellCap >3000 
 
Bauer 1995 
    Cry3Aa crystal >5000 
 
Federici and Bauer 1998 
  Cry1Ba crystal 400 
 
    Cry1Ba protoxin 100 
 
  Cyt1Aa 1.2 
 
L. 
decemlin
eata 
Cry3Aa CC  12 Cry3Aa CellCap 59 
 
Whalon et al. 1993 
  29 Cry3Aa CellCap 293 
 
Utami and Whalon 1995 
C. 
quinquefa
sciatus 
C+O+L+T Cry11A Cq4D 28 11A 42.9 >913** Georghiou and Wirth, 
1997 
    4A/4B 9.3 41.6**  Wirth and Georghiou 
1997  4A/4B/11A 4.7 13.5** 
     4A/4B/11A/Cyt1  2.1 1.1** 
     11A >1000  Wirth et al. 1997 
   11A/Cyt1 7.1 
     11B  9.2 53.1** Wirth et al. 1998 
   11B/Cyt1 7.1 17.5** 
  Cry4A/
Cry4B 
Cq4A
B 
 4A/4B 16.3 >122** Georghiou and Wirth 
1997 
     11A  4.3 >350** Wirth and Georghiou 
1997    4A/4B  2.5 11** 
 
 
 
79
Species Selecting agent Name
c
 Details on Resistance Resistance 
mechanism
f 
 
References 
Type
a
 Form
b
 ES
d
 Formulation/ 
Toxin 
RR
e
 
     4A/4B/11A 10.4 16.2**  
 4A/4B/11A/Cyt1 2.1 3.2** 
     4A/4B 51.1  Wirth et al. 1997 
   4A/4B /Cyt1 0.8 
     11B  9.7 80.7** Wirth et al. 1998 
   11B/Cyt1 1.6 1.6** 
  Cry4A/
Cry4B/ 
Cry11A 
Cq4A
BD 
 4A/4B/11A 13.3 91** Georghiou and Wirth 
1997, Wirth and 
Georghiou 1997 
     11A 18.6 185** Georghiou and Wirth 
1997  4A/4B  5 12.9** 
     4A/4B/11A/Cyt1 1.8 1.2** 
     4A/4B/11A 35.4  Wirth et al. 1997 
   4A/4B/11A/Cyt1 1.3 
     11B 56.2 347** Wirth et al. 1998 
   11B/Cyt1 3.7 3.7** 
  Cry4A/
Cry4B/ 
Cry11A/
Cyt1A 
Cq4A
BD 
Cyt 
 4A/4B/11A/Cyt1 2;3 2** Georghiou and Wirth 
1997 Wirth and 
Georghiou 1997 
     11A 10.4 30.1** Wirth and Georghiou 
1997    4A/4B 5 10.2** 
   4A/4B/11A 5.1 8.1** 
*
This table is adopted from Ferre and van Rie 2002 and literature in this table is updated with research publications after 2002 till 
2006;  Note: Foot note for the table is in next page
80 
 
a
Bta (B. thuringiensis var. aizawai) and Btk (B. thuringiensis var. kurstaki) refer to 
commercial formulations of B. thuringiensis. Dipel, Javelin,Toarow CT and Thuricide are 
tradenames of formulations of Btk. Florbac and Xentari are tradenames for commercial 
formulations of Bta; Bacilex is a tradename for a commercial formulation of a mixture of 
Btk and Bta respectively. Bte = B. thuringiensis var. entomocidus. 
 
b
Different forms of selecting agent have been used: formulated spore-crystal preparations 
(FSC), spore-crystal preparations (SC), micro encapsulated recombinant Pseudomonas 
fluorescens cells expressing a cry gene (CC), inclusion bodies from recombinant 
Escherichia coli cells expressing a cry gene (IB), protoxin (PT) and activated toxin (T). 
 
c
Name of the resistant strain as given in the reference paper, except when followed by '? ': 
these names are arbitrary (or adapted) names given by the authors of this paper. P. 
interpunctella strain 343-R has also been referred to as strain 343. 
 
d
When available, the number of episodes of selection after which the insects were tested, 
is given. 
 
e
RR= resistance ratio; for Bt strains or (pro) toxins this is defined as the LC
50
 (or LD
50
) of 
resistant strain divided by the LC
50
 (or LD
50
) of susceptible control strain, except when 
the value is followed by '*': this values refers to a difference in larval weight between 
resistant and susceptible larvae when larvae were reared on artificial diet with 100 ?g 
toxin/ml. Values in parentheses are estimates. For Bt-plants two values, separated by ?/?, 
are given for the adapted survival on Bt-plants (= % survival on the Bt-plant divided by 
% survival on non-Bt-plants) of the resistant and susceptible strain, respectively. -: not 
determined. All values for Cry1 and Cry9 proteins refer to activated toxins unless 
otherwise indicated. 
 
f 
When available, the mechanism of resistance to the particular toxin is given: reduced 
binding (? binding), slightly altered binding (? binding), unaltered binding (? binding), 
apparently unaltered binding (? binding), increased non-specific binding (? NS binding), 
reduced protoxin activation (? activation), increased toxin degradation (? degradation), 
unaltered proteolytic processing (? processing), increased cell repair or cell replacement 
(? cell repair). In case of binding, only results of binding experiments to native BBMVs 
are given. 
 
81 
 
CHAPTER 2: 
PRODUCTION AND CHARACTERIZATION OF BACILLUS THURINGIENSIS 
CRY1AC-RESISTANT COTTON BOLLWORM, HELICOVERPA ZEA (BODDIE) 
 
Laboratory-selected Bt-resistant colonies are important tools for elucidating Bt 
resistance mechanisms and helping to determine appropriate resistance management 
strategies for Bt crops.  However, some important pest insects such as the cotton 
bollworm, Helicoverpa zea, have proven difficult to select for stable resistance, 
especially when this insect is a pest of both Bt cotton and Bt corn.  Here, two laboratory 
populations of H. zea (AR and MR), resistant to the Bt protein found in all commercial Bt 
cotton varieties in the US (Cry1Ac), were established by selection with either Cry1Ac 
activated toxin (AR) or MVP II (MR).  Cry1Ac toxin reflects the form ingested by H. zea 
when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance 
selection and monitoring. The resistance ratio (RR) for AR reached >100-fold after 11 
generations and has been maintained at this level for 9 generations. This is the first report 
of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations (similar to 
previous observations), reaching only a RR of 12 after 7 generations. AR was only 
partially cross-resistant to MVP II (10% of expected cross resistance) suggesting that 
MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. 
82 
 
zea and that proteases may be involved with resistance.  AR was highly cross-resistant to 
Cry1Ab toxin, but only slightly cross-resistant to Cry1Ab-expressing corn leaf powder.  
AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, 
Vip3A and cypermethrin.  Toxin binding assays showed no significant differences, 
indicating that resistance was not linked to a reduction in binding. These results aid in 
understanding why this major pest of cotton and corn has not yet evolved Bt resistance, 
and highlight the need to choose carefully the form of Bt protein used in experimental 
studies. 
Introduction 
 
Transgenic cotton expressing Bacillus thuringiensis (Bt) Cry1Ac has been used 
commercially in the US since 1995 (10) and the area under Bt cotton production has 
steadily increased over that period (22). Bt cotton provides excellent control of many 
lepidopteran pests of cotton, and thereby exerts tremendous selection pressure for 
resistance.  Concerns regarding resistance to Bt cotton and Bt corn have led the U.S. 
Environmental Protection Agency (EPA) to mandate Insect Resistance Management 
(IRM) strategies for all target pests of Bt crops (11).  Perhaps partly because of these 
IRM strategies, there has yet to be a case of field resistance to Bt cotton after 10 years of 
intense cultivation (4). 
In the US, tobacco budworm, Heliothis virescens F., pink bollworm, 
Pectinophora gossypiella (Saunders), and cotton bollworm, Helicoverpa zea (Boddie), 
are the three major target pests of Bt cotton. Although current Bt varieties express a high 
dose of Cry1Ac against H. virescens and P. gossypiella, it is still not sufficient to kill all 
83 
 
H. zea (21). In particular, high H. zea population pressure and varied expression of 
Cry1Ac in different cotton tissues associated with plant age and stress can result in 
increased H. zea larval survival (1, 19,21). H. zea is highly polyphagous and can be a 
major pest in field corn, and is the key pest in sweet corn in many areas. Therefore, H. 
zea also is exposed to Cry1Ab in Cry1Ab-expressing Bt corn, which is similar in 
structure (>90% amino acid similarity) (8) and mode of action to Cry1Ac. Cross-
resistance to Cry1Ab has been reported in populations of Helicoverpa armigera (2), H. 
virescens (17), H. zea (33), P. gossypiella (45,46,48), and Trichoplusia ni (51).  These 
factors increase the likelihood of resistance development to Bt cotton by H. zea (6, 33). 
Cry1Ac resistance in Bt cotton pests such as H. virescens (17,18) and P. 
gossypiella(45,46,48) is relatively well-studied, using populations selected in the 
laboratory with MVP II (a commercial formulation containing Cry1Ac protoxin inclusion 
bodies encapsulated in Pseudomonas fluorescens cells), and these results have helped 
formulate nationwide IRM strategies. However, these IRM strategies may not be optimal 
for H. zea because Bt resistance mechanisms and other factors can be different in 
different insect species (14, 20). Therefore it is of great interest to establish a Cry1Ac-
resistant H. zea population to examine mechanisms of Bt resistance, patterns of cross-
resistance and other parameters in this insect.  
Several attempts at selecting for Cry1Ac-resistance in H. zea using MVP II have 
been met with limited success (R.E. Jackson, USDA ARS, Stoneville, MS., personal 
communication, WJM unpublished data). Possible reasons for this limited success 
include: fitness costs involved with resistance to the Cry1Ac protoxin or other 
84 
 
compounds in MVP II, or the allele frequency for MVP II resistance being very low (6).  
Furthermore, resistance selection using MVP II may not adequately reflect Cry1Ac 
resistance selection to H. zea in planta because although Bt cotton expresses full-length 
solubilized Cry1Ac protoxin (39), it is at least partially activated to toxin by plant 
proteases immediately upon plant cell disruption. This observation is similar to Cry1Fa in 
cotton, where the full-length protoxin is expressed, but only activated toxin is recovered 
from plant tissue (16).  Besides the use of MVP II, other Cry1Ac forms or preparations 
have also been used for resistance selection including E. coli containing Cry1Ac 
inclusion bodies, Bt Cry1Ac protoxin crystals with spores, and Cry1Ac activated toxin
 
(2,28,33,53). However, it is not known whether there are differences between these 
different Cry1Ac forms in terms of resistance selection as it pertains to Bt cotton. Insect 
susceptibility to Bt proteins may vary with the form of Bt protein ingested, especially in 
Bt-tolerant species, and the form of the Bt protein used may have a dramatic impact on 
the resulting Bt resistance mechanism(s) (26,34,38).  
In this paper, we report for the first time that moderately high and stable 
resistance to Cry1Ac toxin has been attained in H. zea, and this resistance has at least 
been partly characterized.  This strain has differential susceptibilities to various forms of 
Cry1Ac and Cry1Ab, is still susceptible to cypermethrin, and, unlike in most Cry1Ac-
resistant insects, resistance does not appear to be due to alterations in receptor binding 
(14, 20). 
 
MATERIALS AND METHODS 
 
Insect strains: A laboratory susceptible colony of H. zea (SC) was established in 
85 
 
September 2004 from a laboratory colony from Monsanto (Union City, TN). The culture 
at Monsanto is annually infused with insects collected from corn.  Insects were reared on 
pinto bean-based artificial diet at 27 ? 1 
0
C with a photoperiod of 14:10h (L:D) (35). 
Bt proteins and Pyrethroids: An E. coli strain expressing Cry1Ac protoxin from B. 
thuringiensis subsp. kurstaki strain HD-1 (provided by L. Masson, Biotechnology 
Research Institute, National Research Council, Montreal, Canada) was cultured, and the 
activated toxin prepared as indicated elsewhere (34,40). Cry2Aa2 protoxin was prepared 
as described by Moar et al. (34). Cry1Aa (B. thuringiensis EG1273), Cry1Ab (B. 
thuringiensis EG7077) and Cry1Ac (EG11070) clones were provided by Ecogen Inc. 
(Langhorne, Pennsylvania) and were used to prepare trypsin-activated toxins as described 
by Estela et al.(12). MVP II and lyophilized corn leaf powder containing Cry1Ab 
(229.55?g/g) and Cry2Ab2 (6mg/g) were supplied by Monsanto (St. Louis, MO). MVP II 
is a formulated, freeze dried powder containing 19.1% Cry1Ac protoxin inclusion bodies 
encapsulated in Pseudomonas fluorescens. 100% active salt-free Vip3A was supplied by 
Syngenta (Greensboro, NC). A representative pyrethroid, cypermethrin, cyano (3-
phenoxyphenyl) methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate 
(92% a.i. PL86-172) was supplied by FMC Corporation (Philadelphia, PA).  
Selection experiments: Selection experiments were initiated after four generations of 
rearing SC and generating baseline susceptibility values for Cry1Ac toxin and MVP II.  
Two strains of H. zea were selected for Cry1Ac resistance on artificial diet (MR 
and AR, discussed below). Bt protein concentrations were prepared in distilled water and 
mixed thoroughly 20:80 w:w with  artificial diet when the diet temperature was <60 
0
C 
86 
 
and poured onto selection trays. 96 and 384 well micro-titer plates were used as selection 
arenas.  Typically at least 2,000 neonates were used for selection for each of the first 
three generations followed by at least 1,000 neonates for most subsequent selections.  
Individual neonates were exposed to MVP II (MR) or Cry1Ac toxin (AR) for 7d; only 
those larvae that molted (based on larval head capsule size) were selected and reared to 
pupation on diet containing no Bt protein.  
AR was selected at 50 (generation 1), 80 (generation 2-3) and 200 (generation 4-
5) ?g Cry1Ac activated toxin/g. After five generations, selection concentration was 
increased to 500 ?g/g and was not increased further due to limited supply of toxin; for 
every 60 grams of diet, 30 mg of activated toxin was required. AR was selected for 
resistance every generation; currently this strain is under its 25
th
 generation of selection. 
Preliminary experiments showed that MVP II was 2-3 fold less toxic than Cry1Ac toxin.  
Therefore, MR was selected at 100 (generation 1), 200 (generation 2), 500 (generation 3-
5) and 1000 (generation 6-8) ?g of Cry1Ac in MVP II/g diet. Selection of MR could not 
be continued beyond 8 generations due to suboptimal larval number (reduced hatching 
percent), ultimately leading to loss of the strain after generation 11. 
Testing resistance: At selected generations, diet incorporation bioassays were conducted 
concurrently for SC and resistant strains to determine resistance levels. Five to seven 
concentrations of Bt compounds were incorporated into artificial diet as described above 
and assayed against neonates (0 - 16 h old). Each Bt compound-diet concentration was 
poured into 16-32 wells of a 128 well CD International bioassay tray (CD International, 
Pitman, NJ). One ?active? neonate was loaded per well, covered with ventilated covers 
87 
 
(CD International, Pitman, NJ)  and the bioassay trays were incubated at 27 ? 1 
0
C and 
60% RH with a photoperiod of 14:10h L: D. Assays were rated after 7d; dead and first 
instar larvae were considered as dead (3). Bioassays were replicated at least three times. 
Cross-resistance to MVP II: Initial LC
50
 values generated for SC against Cry1Ac 
activated toxin and MVP II at generation 0 indicated a 2.9-fold increase for MVP II 
(Table 1). Based on these observations, similarly higher LC
50
 values for AR were 
expected when tested against MVP II compared to 1Ac toxin. To test this assumption, 
bioassays were conducted with AR using MVP II after 7, 11 and 16 generation of 
selection and concurrently with Cry1Ac toxin. After calculating LC
50
 values from probit 
analysis, the ratios of LC
50 
values for MVP II and activated Cry1Ac toxin were generated 
for AR and SC (Table 2).  
Cross-resistance to other Bt proteins and cypermethrin: Tests for cross-resistance to 
other Cry proteins (Cry1Ab toxin, Cry1Ab-corn powder, Cry2Aa2, & Cry2Ab2-corn 
powder), Vip3A and cypermethrin were conducted between generations 15 and 20 of 
selection (RR about 100-fold). The expression level of Cry1Ab and Cry2Ab2 in corn 
tissue was too low to obtain sufficient mortality; therefore, growth rates on diets 
containing a range of concentrations were used. Mean larval weight was recorded and 
percent weight loss (compared to the untreated control) in different concentrations of Bt 
proteins was calculated considering mean larval weight in untreated control as 100%.  
AR and SC larvae were reared to third instar (8.32 ? 1.29 mg) on untreated diet 
diluted with 20% water and treated topically on the thoracic terga with 0.5 ?l of acetone 
only (control) or 0.5 ?l acetone with a range of cypermethrin concentrations (49). Twelve 
88 
 
larvae were tested per concentration; treated larvae were transferred to 24 well bioassay 
trays containing diet. Additionally, 10 AR larvae (weight: 7.83 ? 1.46 mg) from Cry1Ac 
selection (500 ?g/g of diet) were treated at 1.99 ng/mg body weight. Mortality was 
assessed after 24 h.  All treatments were replicated three times and each replication 
consisted of a total of seven concentrations and a control except as described above. 
Lethal doses were calculated using probit analysis (Polo Plus
?
) and adjusted for body 
weight. 
Labeling of Cry1Ac and Cry1Aa toxins: Cry1Aa and Cry1Ac toxins used for binding 
experiments were obtained from recombinant Bt strains EG1273, and EG11070, 
respectively. Both toxins were trypsin-activated, dialyzed overnight and purified by 
anion-exchange chromatography in a Mono Q HR 5/50 column using an ?KTA explorer 
100x explorer system (GE Healthcare, Uppsala, Sweden) using a 30 ml gradient of 20 
mM Tris-HCl (pH 8.6) to 20 mM Tris-HCl (pH 8.6), 1 M NaCl, as described by Estela et 
al.(12). Sample purity was determined using SDS-PAGE, and protein concentration was 
determined by densitometric analysis using bovine serum albumin as a standard.  
Labeling of Cry1Aa and Cry1Ac was performed by incubating 20 ?g of toxin 
with 0.30 mCi of [
125
I]NaI (Nucliber, Madrid, Spain) using chloramine-T (50). Toxins 
were labeled twice to have relatively fresh labeled-toxins throughout the study. The 
specific activities obtained for Cry1Aa and Cry1Ac were, respectively, 2.3 and 47 
mCi/mg (first labeling) and 0.6 and 1.4 mCi/mg (second labeling). 
BBMV preparation and binding assays: Fifth instar AR and SC larvae were dissected 
in MET buffer (250 mM mannitol, 17 mM Tris-HCl, 5 mM EGTA, pH 7.5) and midguts 
89 
 
were removed and frozen at -80 ?C. Frozen midguts were shipped on dry ice to the 
University of Valencia. Brush border membrane vesicles (BBMV) were prepared by the 
differential magnesium precipitation method (52), frozen in liquid nitrogen and stored at -
80 ?C until used. BBMV protein concentrations were determined by Bradford (5). 
Binding experiments were performed as previously described (12). A fixed 
amount of 
125
I-labeled-toxins and of BBMV (0.05 mg/ml) was incubated for 1 h at room 
temperature with increasing concentrations of unlabeled homologous toxin in 0.1 ml final 
volume of binding buffer (PBS-0.1% BSA: 1 mM KH
2
PO
4
; 10 mM Na
2
HPO
4
; 137 mM 
NaCl; 2.7 mM KCl, pH 7.4, 0.1% BSA). After 10 min 16000 xg centrifugation, pellets 
were washed twice in binding buffer. The final radioactivity remaining in the BBMV 
pellets was measured in a 1282 Compugamma CS gamma counter (LKB, Pharmacia). 
Experiments were replicated twice. N-acetylgalactosamine (GalNAc) was obtained from 
Sigma (St. Louis, Missouri).  Cry1Ac binding in the presence of the GalNac inhibitor was 
performed as described above, but with a pre-incubation of 
125
I-Cry1Ac with GalNAc for 
45 min at room temp., prior to the start of the assay with the addition of the BBMV. 
Experiments were replicated three times. 
Data analysis: Bioassay data were analyzed by probit analysis (15) using POLO-plus 
(LeOra Software, Berkeley, CA, U.S.A.). LC
50
 values with non-overlapping 95% fiducial 
limits were considered as significantly different. Resistance ratios (RR) were calculated 
by dividing the LC
50
 values for AR with that of SC. The percent weight loss data for AR 
and SC in Cry1Ab and Cry2Ab cross-resistance studies was subjected to paired t-tests 
using SPSS (44). A chi square test was conducted to test for significant differences 
90 
 
between ratios of MVP II and Cry1Ac activated toxin LC
50
 values for both AR and SC. 
Binding results were analyzed with the LIGAND computer program (37). 
 
RESULTS 
 
Selection response in AR and MR: There was a significant increase in resistance after 
four generations of selection using Cry1Ac activated toxin (AR) and MVP II (MR) 
compared to the susceptible colony (SC) (Table 1). During the first seven generations, the 
rate of resistance evolution was 3 times faster in AR than in MR. The rate of resistance 
evolution in AR increased with an increase in selection pressure and 12, 36 and 123-fold 
resistance were observed after 4, 7 and 11 generations of selection, respectively (Table 
1). Resistance (based on mortality) in AR did not increase further, as the selection 
concentration was not increased above 500 ?g/g diet due to limited toxin availability, 
however, there has been an increase in the number of large (3
rd
 instar) larvae in 
subsequent generations (KJA, unpublished data). Resistance in MR did not increase 
above 17-fold, even after selecting at higher concentrations for 3 additional generations 
(Table 1). Selection in MR could not be continued beyond 8 generations due to reduced 
larval numbers (lower percentage egg hatch), ultimately leading to loss of the strain after 
11 generations.  
Cross-resistance of AR to MVP II: The ratio of LC
50 
values for MVP II to that of 
Cry1Ac activated toxin for both strains indicated significant (?
2 
= 6.16, p=0.01, df = 1) 
differences (Table 2). Based on these ratios MVP II was more toxic to AR than expected 
91 
 
(should be less toxic, as observed in SC) resulting in only partial cross-resistance (Table 
2).  
Cross-resistance of AR to other Bt proteins and cypermethrin: There was significant 
cross-resistance to Cry1Ab activated toxin (Table 3). AR larvae lost significantly (t 
=14.70, p=0.045) less (11%) weight at the highest concentration of Cry1Ab-expressing 
corn powder (3.84 ?g/g) able to be diet-incorporated (Fig. 1a). There was no cross-
resistance to Cry2Aa2 protoxin inclusion bodies, Cry2Ab2-expressing corn powder (Fig 
1b. t = -0.385, p=0.72), Vip3A and cypermethrin (Table 3). AR was also tested with 
cypermethrin while being reared on diet containing 500 ?g/g of Cry1Ac activated toxin. 
Results (45.8 ? 5.9% mortality at 1.99 ng/mg body weight) were not different when AR 
was reared on regular diet (containing no Cry1Ac).  
Binding of 
125
I-labeled Cry1A toxins to BBMV: Binding of 
125
I-Cry1Ac to brush 
border membrane vesicles (BBMV) from AR and SC did not show significant differences 
even when AR was at its highest resistance ratio. As shown in Fig 2a, homologous 
competition curves followed a similar pattern with BBMV from both strains. Binding 
parameters (dissociation constant, K
d
, and concentration of binding sites, R
t
) obtained 
from the competition experiments were not significantly different (t tests, p>0.05; Table 
4). Binding of Cry1Aa was tested because this toxin shares binding sites with Cry1Ac 
(24) and it has been shown that, in some resistant strains, alteration of a Cry1A common 
binding site may be observed when no differences with Cry1Ac are detected due to 
contribution of other binding sites (29,43). In our case, binding of 
125
I-Cry1Aa did not 
show significant differences between SC and AR (Fig. 2b, Table 4) 
92 
 
To differentiate between binding of Cry1Ac which takes place solely through 
domain II from binding that requires domain III, N-acetylgalactosamine (GalNac) was 
used as a diagnostic tool, as this sugar inhibits binding of Cry1Ac through domain III to 
GalNac residues in the membrane. Preincubation of 
125
I-Cry1Ac with GalNac prior to 
BBMV resulted in partial inhibition of binding (~34%), however, this inhibition was 
similar in both strains (Fig. 3). 
DISCUSSION 
 
Since the advent of transgenic Bt crops, determining the most appropriate form of 
a Bt protein for resistance selection has been an issue for debate. There is a fine balance 
between what forms of the protein(s) are 1) expressed in plants, 2) present in insects upon 
ingestion, 3) available for testing, and 4) the susceptibility of target insects to these 
various protein forms (requiring large quantities of protein if susceptibility is low).  
Historically, and in some cases currently, truncated Bt proteins are expressed within 
transgenic plants, in other cases, full-length protoxins are expressed. However, recent 
reports by Gao et al. (16) and Li et al. (31) demonstrate that what the insect actually 
ingests may be different than what is originally expressed in the plant. As a result, 
researchers are faced with a dilemma of choosing the most appropriate form of the 
protein while facing potential logistical constraints.  Choosing is not an easy task because 
insects can vary in their susceptibility to the various forms of Bt proteins (26). 
Results in this study demonstrate that Cry1Ac-resistant H. zea can be selected and 
maintained using Cry1Ac activated toxin in the laboratory. The laboratory strain 
originating from Monsanto has had annual infusions of H. zea collected from corn and 
93 
 
therefore should have higher genetic variability (and therefore higher Bt resistant allele 
frequency) than laboratory colonies with no infusion of field derived insects. Our initial 
LC
50
 values for SC of 9 ?g/g diet (Cry1Ac toxin) and 26 ?g/g diet (MVP II) are 
significantly higher than those reported by Luttrell et al.(33) for Cry1Ac toxin (0.02 ?g/g 
diet for colony 9103Z) and Ali et al. (3) for MVP II (2.08 ?g/g diet), respectively, for 
their laboratory H. zea colony that has had no infusion of field insects for at least 10 
years. If we compare the LC
50
 of AR at generation 19 to 9103Z, we would observe a RR 
of ~ 69,500.  Although other variables such as bioassay methodology and host strain need 
to be considered, these results suggest that H. zea can be selected to have tremendous 
differences in Cry1Ac susceptibility relative to a highly homogeneous laboratory colony.  
Higher levels of resistance in AR were not sought due to the naturally high tolerance to 
Cry1Ac and cannibalistic nature of H. zea, both resulting in the need for relatively large 
quantities of Cry1Ac activated toxin to rear these insects individually.  The availability of 
appropriate selection materials, especially purified protein and plant material, is still a 
major constraint for producing resistant colonies, especially for those insects that have a 
relatively high tolerance to Bt proteins such as H. zea. 
Resistance development in AR was relatively quick compared to reports for other 
insects
 
(2, 18). Possible reasons for this relatively rapid rate of resistance evolution 
include: selecting only larvae that had molted thereby eliminating a higher percentage of 
susceptible insects in each generation; the use of Cry1Ac activated toxin; and a relatively 
high initial Cry1Ac toxin resistance allele frequency (6).  A relatively rapid rate of 
94 
 
resistance evolution was also observed in S. exigua and another strain of H. zea selected 
using Cry1C and Cry1Ac activated toxin, respectively
 
(33,35). 
The loss of MR after achieving only 17-fold resistance is contrary to reports for 
H. virescens (17,18) and P. gossypiella (46,48). However, our current results with H. zea 
agree with previous unpublished observations by at least two different laboratories. 
Furthermore, concurrent selection with the same parental colony (SC) resulting in 
moderately high and stable resistance to Cry1Ac toxin but not to MVP II further validates 
prior reports. Only partial cross resistance in AR to MVP II suggests further that MVP II 
may not be the most effective Cry1Ac selection agent against H. zea considering that the 
Cry1Ac toxin fragment in MVP II is identical to the Cry1Ac toxin used in selection (8). 
The above statement is based on the following assumptions:  Cry1Ac protoxin (e.g. MVP 
II) is not the only or primary form of Cry1Ac ingested by H. zea when feeding on Bt 
cotton; and the genes necessary to develop resistance to the Cry1Ac protoxin inclusion 
bodies (as in MVP II) were as high in the population as that for Cry1Ac activated toxin 
(6).  
There was a 2.1 to 2.8-fold difference in toxicity between Cry1Ac activated toxin 
and MVP II for SC, and ~2-fold difference would be expected after cleavage of Cry1Ac 
from ~130 kDa to ~65 kDa (35). Therefore, H. zea (SC) does not appear to have 
difficulty converting protoxin to toxin, although potential difficulties could have been 
masked by increased toxicity of other compounds in MVP II (sublethal toxicity to heat-
treated MVP II was observed at the highest rate of MVP II tested in MR selection studies, 
WJM, unpublished data).  MVP II is used to determine H. zea susceptibility in field 
95 
 
populations as part of the EPA-mandated Bt resistance monitoring program. Although the 
precise form and ratio of Cry1Ac toxin and protoxin in Bt cotton is uncertain,  results 
presented here would suggest that the specific methodologies used for determining H. zea 
susceptibility to Cry1Ac in the monitoring program should carefully consider the form of 
Cry1Ac protein used.  Our results would suggest that, if relatively low levels of field 
resistance were to evolve comparable to that which developed in AR, monitoring 
bioassays using MVP II might not be able to identify these resistant individuals. 
Cross resistance studies are invaluable for determining suitable insecticidal 
compounds for pyramiding with Cry1Ac, as well as to help determine possible resistance 
mechanisms.  Current resistance management theory promotes the sequential or 
simultaneous use of different insecticidal compounds provided that cross-resistance does 
not occur among these different toxins (41). As also reported for other Cry1Ac-resistant 
insects (2,17,33,45,46,48,51), AR was cross-resistant to Cry1Ab. This is not unexpected 
because Cry1Ab and Cry1Ac toxins share >90% aa homology (8). However, this cross-
resistance is unlikely to be related to changes in binding affinity of Cry1A toxins because 
no binding differences were observed between SC and AR (24). Only slight cross 
resistance to Cry1Ab corn leaf powder indicates either a possible interaction of Cry 
protein with leaf secondary metabolites or that most/all of Cry1Ab was only partially 
activated and that AR may have difficulty in proteolytically cleaving the protoxin. The 
fact that corn leaf material was immediately freeze-dried after harvesting, and then 
ground into powder, would suggest that plant proteases might have been unable to 
degrade/activate Cry1Ab until after ingestion, indirectly implicating proteolysis as a 
96 
 
potential resistance mechanism. Cry1Ab protoxin activation in corn is further supported 
from a recent study that showed that corn extract partially activated Cry1Ab protoxin, 
suggesting that Cry1Ab protoxin is partially activated by proteases in Bt corn (31) 
Lack of cross resistance to Cry2Aa2 and Cry2Ab2 was probably due to 
differences in aa sequence and mode of action between Cry1Ac and Cry2A (8,9). 
Cry1Ac-resistant H. virescens (strain YHD2), P. gossypiella and H. armigera have 
shown no detectable cross resistance to Cry2A proteins
 
(2,17,46,48). Therefore, our 
results also confirm that the use of Cry2Ab2 pyramided with Cry1Ac (as occurs in 
Bollgard II
?
) should be a viable approach for managing potential resistance to Cry1Ac.  
There was also no cross resistance to Vip3A in AR.  This would be expected because this 
protein does not share any sequence homology with Cry1Ac and is known to bind to 
separate receptors
 
(13,30,54).  Therefore, these results suggest that Vip3A would also be 
a valuable asset in pyramiding Bt proteins for delaying Bt resistance development in H. 
zea (as occurs in VipCot
?
). 
Cypermethrin was tested for cross resistance in AR because growers often spray 
Bt cotton with pyrethroids when high H. zea populations exist, and pyrethroid oversprays 
are currently recommended to mitigate H. zea resistance to Bt cotton (21). AR was tested 
with cypermethrin both on untreated and Cry1Ac-treated diet.  The primary reasoning 
behind the use of Cry1Ac-treated diet was to more realistically simulate pyrethroid 
exposure to a potentially Bt-resistant H. zea larva feeding on Bt cotton. Because no cross-
resistance to cypermethrin was observed for larvae feeding either on untreated or 
Cry1Ac-treated diet, these results would suggest that pyrethroids can continue to be used 
97 
 
when necessary, and probably have been a valuable Bt cotton IRM practice since the 
introduction of Bt cotton in 1996.  
The narrow spectrum of Bt resistance suggests an alteration in the binding site of 
Cry1Ac (14). However, in contrast to other Cry1Ac resistant insects, we did not detect 
any significant reduction in binding. Lack of Cry1Ac binding has been reported in some 
Cry1Ac resistant populations of H. virescens (23), P. gossypiella (36), H. armigera (2), 
P. xylostella (42, 47) and T. ni (51). Because Cry1Ac is known to bind to GalNac 
residues of glycosylated membrane proteins (27), we tried to dissect Cry1Ac binding 
using GalNac as an inhibitor, thus discriminating between GalNac-dependent and 
GalNac-independent binding (12). Again, we could not find any binding difference 
between both strains. Another way to look for binding alterations is to use different 
Cry1A toxins known to bind to a common receptor. Cry1Aa, Cry1Ab and Cry1Ac share 
binding sites in H. zea (24). Although Cry1Aa has low toxicity to H. zea, this toxin was 
used in binding analyses as a diagnostic tool because it has been shown that in H. 
virescens and Ostrinia nubilalis, resistant insects that showed reduced or no binding of 
Cry1Aa to the Cry1A common receptor still could bind Cry1Ac (29,43). Similar to 
Cry1Ac, there were no significant differences in Cry1Aa binding in terms of either 
dissociation constants (K
d
) or concentration of binding sites (R
t
) for Cry1Aa among the 
samples. Therefore, reduction in binding does not seem to be the mechanism of resistance 
in AR, in spite of the narrow spectrum of cross-resistance observed. The fact that total 
cross-resistance does not even extend to protoxin forms of Cry1Ac (MVP II) and Cry1Ab 
(Bt-corn powder), might be indicative of a differential activation of protoxin in the insect 
98 
 
midgut, as opposed to in vitro bovine-trypsin activation (25, 38). Alternatively, the C-
terminal end of the protoxin may protect the active toxin from the degradative action of 
midgut proteases, resulting in a higher yield of the fully active toxin (7, 32). 
Results from this study demonstrate that broad assumptions cannot be made that 
all target pests will respond in the same manner to a particular Bt (protein or 
formulation). Because AR represents just a single strain, additional selections against 
geographically distinct H. zea populations are recommended to determine potential 
different resistance characteristics, as has been demonstrated for H. virescens (17,18). 
Although AR is currently only about 100-fold resistant to Cry1Ac, we feel that this level 
of resistance is appropriate for characterization because:  H. zea is 10-40 fold less 
susceptible to Cry1Ac than H. virescens or P. gossypiella; a lower level of resistance 
necessary to survive on Bt cotton might be expected and lower levels of Bt resistance not 
resulting in total survivorship on Bt cotton might be appropriate for initiating alternative 
control strategies, and higher levels of resistance are difficult to achieve due to logistical 
constraints. We have shown that H. zea does react differently to Cry1Ac activated toxin 
and MVP II than other cotton pests, and therefore this information can be used to more 
adequately adopt cotton IRM strategies for all target pests.  Possible implications could 
include: If H. zea has difficulty evolving resistance to full-length or mature forms of Bt 
proteins (as suggested for MVP II), proteins could be designed appropriately; if 
resistance is not primarily due to binding differences, other potential resistance 
mechanisms should be explored.  Our results also show that Cry1Ac-resistant H. zea is 
susceptible to Cry2Ab2 (found in Bollgard II
?
), Vip3A (found in VipCot
?
), and 
99 
 
pyrethroids such as cypermethrin.  These results show that the cotton growing 
community has many alternative control methods to help delay the evolution of Cry1Ac 
(and other Bt proteins) resistance for the future. 
 
Acknowledgements 
 
The authors thank Nancy Adams, Monsanto Co., Union City, TN for providing H. zea; 
Monsanto Co. for providing Cry1Ab, Cry2Ab2 and Syngenta for providing Vip3A; and 
G. Head, Monsanto Co., Saint Louis, MO for review of this manuscript.   
This research was supported by USDA, Cotton Incorporated and by the Spanish 
Ministry of Education and Science (projects No. AGL2003-09282-C03-01 and 
AGL2006-11914). 
 
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47. Tabashnik, B.E., Y. B. Liu, T. Malvar, D. G. Heckel, L. Masson, V. Ballester, F. 
Granero, J. L. Mensua and J. Ferre. 1997. Global variation in the genetic and 
biochemical basis of diamondback moth resistance to Bacillus thuringiensis. Proc. 
Natl. Acad. Sci. USA 94:12780-12785. 
48. Tabashnik, B.E., Y. Liu, R. A. de Maagd and T. J. Dennehy. 2000. Cross-resistance 
of pink bollworm (Pectinophora gossypiella) to Bacillus thuringiensis toxins. 
Appl. Environ. Microbiol. 66:4582-4584. 
49. Usmani, K.A. and C. O. Knowles. 2001. Toxicity of pyrethroids and effect of 
synergists to larval and adult Helicoverpa zea, Spodoptera frugiperda, and 
Agrotis ipsilon (Lepidoptera: Noctuidae) J. Econ. Entomol. 94:868-873  
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50. Van Rie, J., S. Jansens, H. Hofte, D. Degheele and H. Van Mellaert. 1989. Specificity 
of Bacillus thuringiensis ?-endotoxins. Importance of specific receptors on the 
brush border membrane of the midgut of target insects. Eur. J. Biochem. 186:239-
247. 
51. Wang, P, J. Z. Zhao, A. Rodrigo-Simon, W. Kain,  A. F. Janmaat, A. M. Shleton, J. 
Ferre and J. Myers. 2007. Mechanism of resistance to Bacillus thuringiensis toxin 
Cry1Ac in greenhouse population of cabbage looper, Trichopusia ni. Appl. 
Environ. Microbiol. 73:1199-1207. 
52. Wolfersberger, M.G., P. Luethy, P. Maurer,  P. Parenti, V. F. Sacchi, B. Giordana,  
and G. M. Hanozet. 1987. Preparation and partial characterization of amino acid 
transporting brush border membrane vesicles from the larval midgut of the 
cabbage butterfly (Pieris brassicae). Comp. Biochem. Physiol. 86A: 301-308. 
53. Xu, X., L.Yu, and Y. Wu. 2005. Disruption of a cadherin gene associated with 
resistance to Cry1Ac delta -endotoxin of Bacillus thuringiensis in Helicoverpa 
armigera. Appl. Environ. Microbiol. 71:948-954. 
54. Yu, C., M. A. Mullins, G. W. Warren, M. G. Koziel and J. J. Estruch. 1997. The 
Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut 
epithelium cells of susceptible insects. Appl. Environ. Microbiol. 63:532-536. 
108 
 
Table 1. Resistance development in H. zea when selected using activated toxin and  
MVP II 
Strain G
a
 N
b
 LC
50
 (95% FL)
c
 Slope (mean ? SE) RR
d
 
Activated toxin-resistant strain (AR) 
SC
e
  800 9.13 (5.83- 12.53) 1.61? 0.41  
AR 4 384 107.64 (75.37 ? 155.6) 1.42 ? 0.3 12.12 
SC  480 8.89 (5.71-13.72) 1.71? 0.41  
AR 7 222 321.22 (251.27 ? 371) 1.89 ? 0.13 35.91 
SC  384 8.94 (6.37 ? 15.27) 1.93 ? 0.31   
AR 11 175 1,450 (690 ? 2,392) 1.42 ? 0.47 122.67 
SC  384 11.82 (7.01-19.24) 1.76 ? 0.42  
AR 16 72 47% survivors @ 1.5mg/g  >100 
SC  384 13.90 (9.11- 21.44)  2.41 ? 0.51  
AR 19 192 1,390 (743 ?12,017) 1.39 ? 0.46 92.69 
SC  192 15.00 (9.90 ? 22.45) 2.31 ? 0.52  
MVP II resistant strain (MR) 
SC 0 1120 26.13 (16.34 ? 35.62) 1.73 ? 0.24  
MR 4 384 384.3 (282.31 ? 568.12) 1.79 ? 0.27 16.61 
SC  640 23.13 (16.34 ? 35.62) 1.73 ? 0.24  
MR 7 672 298.40 (155.16 ? 455.5) 1.67 ? 0.41 12.01 
SC  1120 24.84 (13.48 ? 41.89) 2.47 ? 0.57   
MR 9-11 No selection due to reduced larval number, resistant strain crashed after 
11 generations 
 
109 
 
 
a
Generations of H. zea continuously selected with Bt  
b
Total number of insects tested (one to five replicates with one to seven concentrations). 
c
LC
50 
values are in micrograms of Bt protein per gram of diet. FL, fiducial limits 
d
Resistance ratio: LC
50
 for AR divided by the LC
50
 for SC 
e
SC: Susceptible colony 
   
110 
 
Table 2. Cross-resistance of AR to MVP II 
Strain G
a
 N
b
 LC
50
 (95% FL)
c
 Slope  
(mean ? SE) 
RR
d
 MVP II ? 
1Ac Ratio
e
 
AR 7 (36) 192 117.79(63.38 ? 175.45) 1.81 ? 0.34 4.7 0.37 
SC
f
  1120 24.84 (13.48 ? 41.89) 2.47 ? 0.57   2.78 
AR 11 (123) 576 197.10 (134.1 ? 333.51) 1.36 ? 0.29 7.9 0.14 
SC  576 24.94 (13.86 ? 44.87) 2.57 ? 0.37  2.11 
AR 16 (>100) 448 397.93 (245.87 ? 749.86) 1.56 ? 0.42 10.3 0.28 
SC  448 38.53 (24.87 ? 55.56) 2.41 ? 0.53  2.76 
a
Generations of H. zea continuously selected with Cry1Ac activated toxin; Values in 
parenthesis indicate resistance ratio to Cry1Ac activated toxin. 
b
Total number of insects tested (one to five replicates with one to seven concentrations). 
c
LC
50
s are in micrograms of Cry1Ac in MVP II per gram of diet. FL, fiducial limits 
d
Resistance ratio: LC
50
 for AR divided by the LC
50
s for SC 
e
MVP II-1Ac
 
Ratio. Ratio of LC
50
 values for MVP II divided by the LC
50
 values for 
Cry1Ac activated toxin (data from Table 1) 
f
SC: Susceptible colony 
 
111 
 
Table 3. Cross-resistance of AR to other B.  thuringiensis proteins and cypermethrin 
Strain G
a
 Compound N
b
 LC
50
 (95% FL)
c
 Slope  
(mean ? SE) 
RR
d
 
AR 19 (93) Cry1Ab 200 8.44 % mortality @ 400?g/g of diet ND
e
 
SC
f
   200 133.33 (98.42 ? 261.41) 1.82 ? 0.55  
AR 15 (>100) Vip3A 512 22.29 (15.18 ? 31.07) 2.59 ? 0.49 0.94 
SC   512 23.73 (16.82 ? 33.80) 2.24 ? 0.35  
AR 16 (>100) Cry2Aa2 672 101.83 (72.60 ? 167.39) 2.83 ? 0.51 1.55 
SC   672 65.70 (46.27 ? 109.34) 1.89 ? 0.27  
AR 16 (>100) Cypermethrin 288 1.70 (1.11 ? 2.61)
g
 2.08 ? 0.40 1.85 
SC   288 0.92 (0.61 ? 1.33)
g
 2.55 ? 0.52  
a
Generations of H. zea continuously selected with Bt protein; values in   
  parenthesis indicates resistance ratio when bioassays were conducted 
b
Total number of insects tested (three to five replicates with five to seven concentrations). 
c
LC
50
s are in micrograms of Bt protein per gram of diet. FL, fiducial limits  
d
Resistance ratio: LC
50
 for AR divided by the LC
50
s for SC    
e
ND: Not determined because LC
50
 for AR could not be obtained 
f
SC: Susceptible colony 
g
Lethal dose (ng/mg body weight) 
 
112 
 
Table 4. Dissociation constants (K
d
) and concentration of binding sites (R
t
) for binding of  
              Cry1A proteins to BBMV from H. zea
a
. 
Bt toxin Sample (gen. selection) K
d
 ? SD (nM) R
t
 ? SD (pmol/mg) 
b
 
Cry1Ac                SC
c
 1.1 ? 0.1 17.0 ? 0.9 
 AR
c
 (4) 0.50 ? 0.3 22.3 ? 4.9 
                AR (7) 0.4 ? 0.1   28 ? 15 
 AR (11) 2.9 ? 0.1 49 ? 3 
Cry1Aa                SC 3.2 ? 0.4  2.7 ? 0.7 
                AR (7) 3.8 ? 0.1  4.1 ? 0.7 
 AR (11) 3.2  ? 0.3  5.0 ? 1.2 
a
Values are the mean of two replicates for resistant insects and four replicates for the SC 
strain (using two independently labeled Cry1Ac and Cry1Aa batches) 
b
Expressed as pmol per milligram of total vesicle protein 
c
SC: Susceptible colony; AR: Cry1Ac-resistant colony 
 
 
113 
 
Fig 1. Toxicity of Cry1Ab (a) and Cry2Ab2 (b) expressing corn leaf powder to 
susceptible (SC) and Cry1Ac-resistant (AR) H. zea. Values are expressed as percentage 
weight loss relative to the corresponding controls at different concentrations. Data 
represent the mean of four replications and error bars are the standard deviation. The 
asterisk (*) indicates significant differences in t-tests (t =14.70, p=0.045) 
Cry1Ab (?g/g of diet)
1.92 3.84
P
e
r
c
e
n
t
a
ge
 w
e
i
ght
 l
o
s
s
0
20
40
60
80
100
SC 
AR 
 
Cry2Ab2 (?g/g of diet)
3.13 6.25 12.50 25.00 50.00100.00
P
e
r
c
ent
age
 w
e
i
g
h
t
 
l
o
ss
0
20
40
60
80
100
SC 
AR 
 
 
b
*
a
114 
 
Fig 2. Binding of 
125
I-Cry1Ac (A) and 
125
I-Cry1Aa (B) to BBMV from susceptible (SC) 
(z) and and Cry1Ac-resistant (AR) H. zea (S) at increasing concentrations of unlabeled 
homologous competitor. Data represent the mean of two experiments and error bars are 
the SEM. 
 
0
0
20
40
60
80
100
10
-1
10
0
10
1
10
2
10
3
[Cry1Ac] (nM)
125
I
-
C
r
y1A
c
 ini
t
i
a
l
 bin
d
in
g (%
)
A
 
 
0
40
60
80
100
10
-1
10
0
10
1
10
2
10
3
[Cry1Aa] (nM)
125
I-C
ry1A
a init
ial bindi
ng 
(%
)
B
 
 
a
b
115 
 
Fig 3. Percent binding of 
125
I-Cry1Ac to BBMV from susceptible (SC) and Cry1Ac-
resistant (AR) H. zea in the absence (SC and AR) or the presence (LC+GN and AR+GN) 
of 25 mM GalNac. Data represent the mean of three experiments and error bars are the 
SEM. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AR 
AR + GN 
0
25
50
75
100
125
%
125
I
-
C
r
y
1
A
c
 bi
nding
SC + GN SC 
116 
 
CHAPTER 3: 
FITNESS COSTS ASSOCIATED WITH CRY1AC-RESISTANT HELICOVERPA 
ZEA (LEPIDOPTERA: NOCTUIDAE): A FACTOR COUNTERING SELECTION 
FOR RESISTANCE TO BT COTTON? 
 
ABSTRACT The heritability, stability, and fitness costs in a Cry1Ac-resistant 
Helicoverpa zea colony (AR) were measured in the laboratory.  In response to selection, 
heritability values for AR increased in generations 4 to 7 and decreased in generations 11 
to 19.  AR had significantly increased pupal mortality, a male-biased sex ratio, and lower 
mating success compared to the unselected parental strain (SC). AR males had 
significantly more mating costs compared to females.  AR reared on untreated diet had 
significantly increased fitness costs compared to rearing on Cry1Ac treated diet.  AR had 
significantly higher larval mortality, lower larval weight, longer larval developmental 
period, lower pupal weight, longer pupal duration, and higher number of morphologically 
abnormal adults compared to SC.  Due to fitness costs after 27 generations of selection as 
described above, AR was crossed with a new susceptible colony (SC1), resulting in AR1.  
After just two generations of selection, AR1 exhibited significant fitness costs in larval 
mortality, pupal weight and morphologically abnormal adults compared to SC1.  
Cry1Ac-resistance was not stable in AR in the absence of selection.  This study 
117 
 
demonstrates that fitness costs are strongly linked with selecting for Cry1Ac resistance in 
H. zea in the laboratory, and fitness costs remain, and in some cases, even increase after 
selection pressure is removed.  These results support the lack of success of selecting, and 
maintaining Cry1Ac-resistant populations of H. zea in the laboratory, and may help 
explain why field-evolved resistance has yet to be observed in this major pest of Bt 
cotton. 
Introduction 
The evolution of resistance in target insect populations is the primary concern with the 
use of crops expressing Bacillus thuringiensis (Bt) proteins such as Cry1Ac in Bt cotton 
(Bollgard
?
) in the US and elsewhere.  However, even after 12 years of commercial use in 
the US, there are still no documented cases of field-evolved resistance in Bollgard
? 
to any 
of the three target pests, especially to bollworm, Helicoverpa zea (Boddie) (Moar and 
Anilkumar 2007). H. zea is significantly more tolerant to Cry1Ac present in Bollgard
? 
than other target pests (MacIntosh et al. 1990, Ali et al. 2006, Sivasupramaniam et al. 
2008) and can survive on Bollgard
? 
late season (Jackson et al. 2004a).  Although 
resistance management strategies such as ?high dose plus refuge? have been used to 
delay resistance development (Gould 1998) the use of these tactics alone cannot fully 
explain the total lack of field-evolved resistance. Factors such as fitness costs, stability, 
and the genetics of resistance may play a significant role in delaying or mitigating 
resistance evolution (Tabashnik 1994, Gould 1998).  Many models have predicted the 
delay in resistance development due to fitness costs (Caprio 2001, Storer et al. 2003a, 
2003b; Gustafson et al. 2006). Studies with laboratory-selected Cry1Ac-resistant insects 
118 
 
such as Pectinophora gossypiella (Saunders), Helicoverpa armigera (Hubner), and 
Plutella xylostella (L.) support these model predictions by documenting fitness costs and 
incomplete resistance to Bt crops (Liu et al. 1999, Carri?re et al. 2001a, 2001b, 2006; 
Sayyed and Wright 2001, Bird and Akhurst 2004, 2005; Higginson et al. 2005).  
Selection for Cry1Ac-resistant H. zea in the laboratory has been attempted 
numerous times over a 10 year period, but all attempts have resulted in colony crashes 
due to fitness costs (Luttrell et al. 1999, Luttrell R, Univ. of Arkansas, personal 
communication, Jackson et al. 2004b, Jackson, R., USDA, personal communication., 
WJM unpublished data, Anilkumar and Moar 2006, Anilkumar et al. 2008).  
Additionally, although there have been many attempts to rear field-collected H. zea 
populations, often collected from Bt crops, with relatively high tolerances to Cry1Ac 
(MVP II) in the laboratory, they typically cannot be maintained for more than five to 
seven of generations, with many populations crashing after one to two generations 
(Luttrell, R, Univ. of Arkansas, personal communication, KJA and WJM, unpublished 
results).  Anilkumar et al. (2008) reported a stable Cry1Ac-resistant H. zea strain (AR) 
after selection with Bt Cry1Ac toxin for 11 generations (100-fold resistance) and this 
colony was maintained at this level for 25 generations under continuous selection.  Even 
though AR was relatively stable, fitness costs were observed during selection and when 
insects were removed from selection. (Note:  This colony was crossed to a susceptible 
population in Generation 26 to avoid the total collapse of this strain due to fitness costs).  
Further, higher fitness costs usually affect the stability of resistance in the population, 
thereby affecting the heritability of resistance.  Therefore, a thorough understanding of 
119 
 
the biological traits of H. zea affected by fitness costs, heritability and stability of 
resistance could contribute to the development of more realistic models for predicting the 
development of resistance and thereby aid in formulating better strategies for effective 
resistance management.  Furthermore, findings in this paper could help explain why 
field-evolved resistance has yet to be observed in this pest to Bollgard
?
 after 12 years of 
intense use.  Therefore, this study investigated the various biological parameters 
associated with fitness costs, heritability and stability of resistance exhibited by Cry1Ac 
toxin-resistant H. zea (Anilkumar et al. 2008).  
Materials and methods 
Insect strains: A laboratory susceptible colony of H. zea (SC) was established in 
September 2004 from a laboratory colony from Monsanto (Union City, TN).  The culture 
at Monsanto was annually infused with field-collected insects; therefore, the population 
was heterogeneous and contained Cry1Ac-resistant genes (Anilkumar et al. 2008).  One 
strain (AR) was selected from SC for resistance on artificial diet containing Bt Cry1Ac 
toxin for 25 generations by exposing individual neonates for seven days (Anilkumar et al. 
2008).  Only second and third instars were transferred to 24 well tissue culture plates 
containing untreated diet and were reared an additional seven days (Ali et al. 2006, 
Anilkumar et al. 2008, Sivasupramaniam et al. 2008). Late 4
th
 to early 5
th
 instar larvae 
were transferred to diet cups (30 ml, Bio-serve, Frenchtown, NJ) containing artificial diet 
and were reared to pupation.  Except for selection using Cry1Ac toxin, AR and SC were 
treated similarly in regards to diet used, number of larvae reared, quality of larvae 
harvested, and number of adults used for generating subsequent generations.   
120 
 
Resistance heritability and resistance risk assessment: Heritability of resistance (h
2
) 
and resistance risk (G) (number of generations required for 10-fold increase in resistance) 
were estimated.  LC
50
 values for SC and AR conducted simultaneously (Anilkumar et al. 
2008) and percent survival in each generation of selection were used for calculating 
parameters necessary for determining h
2
 and G (Tabashnik 1992).   
Fitness costs in AR on Cry1Ac treated diet: While conducting selection experiments, a 
reduction in egg hatch was observed in AR after nine generations of selection (>36-fold 
resistance, Anilkumar et al. 2008).  Further observations indicated no embryo 
development, confirming egg infertility.  Therefore, both resistant (AR) and control (SC) 
strains were monitored for mating success during resistance selection, and maintenance, 
respectively, from generations 9-24. 
Larvae were selected and reared as discussed above; the resistance ratio of AR 
(LC
50
 of AR / LC
50
 of SC) exceeded 100-fold.  The resulting pupae were sexed and 
maintained in separate boxes (18x18x7cms) for adult eclosion.  Pupal sex ratio was 
recorded for 15 generations (generations 10 to 24; 6,314 pupae total).  Further, pupal 
mortality (dead pupae and malformed adults) were recorded for 11 generations 
(generation 15 to generation 25; 4,867 pupae total).  The proportions of males were 
modeled as the number of males in a total population, and were analyzed using a 
binomial test; the interaction of population and generation were considered as residuals 
(Hardy 2002, SAS Institute 2003).  Pupal mortality was compared between strains using 
a paired t-test (SPSS 2006).  
121 
 
Initially adults were released into mating cages (34x19x11cms) at a 1:1 sex ratio 
and thirty moths were maintained per cage. However, additional (maximum of three) 
moths from either sex were released into mating cages because of premature adult 
mortality in some of the original 30 moths (within 3 three days).  The resultant sex ratio 
was not significantly different (see results below) from 1:1.  Mating cages were covered 
with white cloth for oviposition and moths were fed a 10% sucrose solution.  Egg sheets 
were replaced daily and incubated at 27 ? 2 
0
C until hatching.  Adults were maintained in 
cages until death or when moths quit laying eggs (after 10 days).  Dead moths were 
removed daily from cages, and all surviving moths (after 10 days) were dissected under a 
stereo microscope to determine mating frequency.  Female moths were classified as 
mated or unmated based on the presence or absence of spermatophore(s) in the 
spermatheca.  Further, females were classified as having mated once, twice, three, four or 
five times depending on the number of spematophores present in the spermatheca.  Mean 
number of spermatophores produced per male was calculated by taking the total number 
of spermatophores produced in a generation divided by the number of males released into 
cages (Bird and Akhurst, 2004).  Observations were made for 14 generations (generation 
9 [Resistance Ratio {RR} >36-fold], and generations 12 to 24 [RR >100-fold]) from a 
period spanning nearly two years and from a total of 3,886 moths.  Percentage mating, 
multiple mating and mean number of spermatophores between AR and SC were 
compared by paired t-tests (SPSS 2006). 
Mating propensity observations indicated a reduction in mating success in AR 
(see results, Table 2; Fig. 2).  Therefore, reciprocal crosses between AR and SC (AR[?] 
122 
 
X SC[?] and SC[?] X AR[?]) were conducted at an equal sex ratio to test if the 
reduction in mating success was sex-linked.  Moths were caged and percent mating was 
ascertained as described above. Reciprocal crosses were conducted with five male and 
five female moths spanning three generations, >50 moths (1:1 sex ratio) in two 
generations (two replicates). Therefore, the total number of moths used in each of the two 
reciprocal (AR X SC) crosses was 140.  Percentage mating, multiple mating and mean 
number of spermatophores between AR and SC were analyzed by ANOVA (SPSS 2006). 
Fitness costs in AR on untreated diet: The relative performances of both susceptible 
(SC) and >100-fold resistant (AR) strains were measured on untreated artificial diet 
(referred to hereafter as regular diet) and untreated selection diet (artificial diet diluted 
with 20% water; used for incorporating Bt proteins in selection experiments, referred to 
hereafter as selection diet) (Anilkumar et al. 2008).  A total of 160 larvae (48, 48 and 64 
larvae in replication 1, 2 and 3, respectively) for each treatment were tested.  Individual 
neonates were placed on diet in 128 well CD International bioassay trays (CD 
International, Pitman, NJ) and reared for seven days.  Larval weight and instar were 
recorded after seven days, and larvae were transferred to 30 ml diet cups containing 
regular diet and reared until pupation.  Larval duration and mortality were recorded.  All 
insects were removed from diet on the second day of pupation; weights were recorded 
and were transferred to a new 30 ml cups (containing no diet).  Pupae were sexed and 
observed daily for adult eclosion.  Adults failing to eclose and those with fringed wings 
were considered as malformed adults.  Pupa that did not eclose after 15 days was 
considered dead. 
123 
 
Growth rate (weight gain per day) was calculated for both strains after the first 
seven days (on either diet) and at pupation.  The growth rate for the first seven days 
(when insects were exposed to either selection diet or regular diet) was calculated by 
dividing larval weight by seven.  The growth rate after 7 days, (when insects with 
different exposure background were transferred to regular diet) was calculated by the 
following equation.  
Growth rate (weight gain per day) = (Pupal weight ? larval weight at 7 days) 
                                     (Larval duration ? 7 days) 
 
Further, the difference in growth rates was calculated by subtracting the growth rate 
during the first seven days from that determined after seven days.  Insects that died 
prematurely were not included in the analysis.     
Thirty adults were released into mating cages and maintained as explained above.  
Total number of eggs laid was recorded daily and mean number of eggs per female was 
calculated.  Eggs were incubated for 4 days at 27 ? 2 
0
C, and hatching percentage was 
calculated.  Each experiment on selection and regular diets was considered as a block, 
each insect as a replicate and the entire test as a randomized complete block design for 
analysis.  Larval and pupal periods were log transformed to stabilize variance. Larval 
weight, duration and mortality; pupal weight and duration; and percent malformed adults 
were analyzed using two-way ANOVA and means were separated using Tukey?s least 
significant differences (SPSS 2006).  Growth rates during initial seven days, after seven 
days, and their difference were analyzed using ANOVA and means were separated using 
Fisher least significant differences (SPSS 2006).   
124 
 
Crosses with the susceptible strain: AR was crossed with a new susceptible strain 
(SC1) resulting in AR1, to avoid complete loss of the strain due to fitness costs (see 
results) associated with Cry1Ac-resistance selection and maintenance.  As discussed 
earlier, the laboratory colony at Monsanto is infused annually with field collected insects; 
therefore, SC1 is a derivative of SC from the most recent infusion in 2007.  SC1 had 
increased tolerance to Cry1Ac toxin (LC
50
=31.25?g Cry1Ac/g of diet) compared to SC 
(LC
50
=9-15?g Cry1Ac/g of diet, Anilkumar et al. 2008).  Even though both reciprocal 
crosses were attempted, only AR[?] X SC1[?] yielded a F
1
 population due to mating 
costs associated with AR males (see results).  AR1 was selected at the regular selection 
concentration of Cry1Ac (500 ?g Cry1Ac/g of diet) for two generations.  Further, fitness 
parameters (discussed above) were measured only on regular diet.  Three experiments 
were conducted with 32 larvae each per strain per replication and data were analyzed as 
discussed above.   
Stability of resistance: The desired number of larvae could not be obtained for bioassays 
when AR was reared on untreated diet for two generations due to extremely high pupal 
mortality (discussed in Results). Therefore, bioassays were conducted immediately after 
one generation.  Neonates (130 in number) were tested in two replications at 500 ?g 
Cry1Ac/g of diet (concentration used in resistance selection/maintenance experiments) 
compared with an untreated control. Parallel tests were conducted on AR subjected to 
continued selection.  Paired t-tests were conducted to compare the survivorship of AR on 
500 ?g Cry1Ac/g of diet when AR was continuously selected at 500 ?g Cry1Ac/g of diet, 
and after AR had been reared one generation on regular diet.  
125 
 
 Statistical analysis:  All statistical tests were conducted at the 0.05 level of significance 
using either SPSS or SAS statistical programs, and for those parameters which required 
transformations for stabilizing the variance, data are presented as non-transformed 
arithmetic means.   
Results 
Heritability and resistance risk assessment: The heritability (h
2
) of resistance to Bt 
Cry1Ac toxin varied at different generations of selection (Table 1).  The h
2
 was 0.315 
after four generations (12-fold resistance), increased to 0.401 after seven generations (36-
fold resistance) and decreased to 0.256 and 0.123 after 11 and 19 generations, (>100-fold 
resistance) respectively.   Resistance risk (G) assessment considering heritability values 
after 19 generations of selection (0.123) indicated that 9.66 generations are required for a 
10-fold increase in resistance. 
Fitness costs in AR on Cry1Ac treated diet: 
Pupal sex ratio and mortality: There were significant (F
1,14
=9.44, p=0.0083) 
differences in sex ratios of AR and SC (Fig 1).  In 11 of 15 generations AR produced 
more males than females as compared to only 3 of 15 generations of male bias in SC.  
Results from 11 generations indicated that mean (? SE) pupal mortality in AR (24.48 ? 
2.47%) was significantly (t
10
=5.244, p<0.001) higher than SC (11.67 ? 1.16%). However, 
there were no significant differences in mortality between sexes for either AR (t
10
=-
1.138, p=0.284) or SC (t
10
=-0.881, p=0.401).  
Mating studies: Percent mating success during generations 9 and 12 was not 
significantly (t
1
= -1.963, p=0.30) different between AR and SC (Fig. 2).  Resistance 
126 
 
ratios for these generations were 36-fold and 122-fold, respectively (Anilkumar et al. 
2008).  Mating success in AR declined after achieving >100-fold resistance and there was 
always a 1.5 to 3-fold decrease in mating success for AR compared to SC.  Further, 
significantly (t
13
=-2.521, p=0.026) more SC females (26%) had multiple mating 
compared to AR (17%) (Table 2).  SC males (1.23 ? 0.10) produced significantly (t
13
=-
5.058, p=0.001) more spermatophores compared to AR (0.58 ? 0.08).   
Reciprocal crosses:  Mating success between AR and SC was significantly (F
3,19
=14.29, 
p=0.000) different (Table 3).  Reciprocal crosses (AR? X SC?, SC? X AR?) with SC 
as male had significantly (p=0.046) higher mating compared to AR as male. There were 
no significant differences in mating between AR and reciprocal cross with AR as male 
(p=0.35); similarly between SC and SC as male in reciprocal cross (p=0.61).  There were 
no significant differences in multiple mating between either parental strains or their 
reciprocal crosses (F
3,19
=0.7, p=0.566). The number of spermatophores produced per 
male was significantly (F
3,19
=3.804, p=0.028) different in AR and SC strains.  However, 
when AR males were used in the reciprocal cross, no significant differences were 
observed in mean spermatophore/male compared with SC as male (p=0.074) and/or SC 
strain (p=0.099).   
Fitness costs in AR on untreated diet:   
Larval weight, duration and mortality: SC gained significantly (F
3,574
=48.178, 
p=0.000) more weight in seven days compared with AR.  Further, selection diet or 
regular diet did not affect larval weight in SC (Table 4).  In contrast, AR on selection diet 
had significantly lower weight compared to when reared on regular diet. Significant 
127 
 
differences (F
3,571
=124.01, p=0.000) existed between strains for larval duration regardless 
of diet tested; AR required one additional day to complete larval development compared 
with SC.  Further, rearing larvae either on selection diet or regular diet for one week did 
not influence the total larval duration in either AR or SC.  It is important to note that 
significantly (F
3,11
=4.623, p=0.037) higher larval mortality was recorded in AR compared 
to SC, although larval mortality did not differ between selection and regular diet in either 
AR or SC.  
Pupal weight, duration and mortality: Pupal weight of AR on regular diet varied 
significantly (F
3,503
=25.402, p=0.000) from SC.  Interestingly, AR pupal weight on 
selection diet was not different from SC.  SC on regular diet recorded the shortest pupal 
duration which was significantly (F
3,492
=39.425, p=0.000) different from SC on selection 
diet and AR on both diets.  Production of morphologically abnormal adults in AR was 
significantly (F
3,11
=14.281, p=0.001) increased (ca 6-fold) when compared with SC, 
which did not differ between selection diet and regular diet and had the most pronounced 
effect on fitness in relation to resistance.  
Growth rate: During the initial seven days, weight gained per day by SC larvae was 
significantly (F
3,471
=22.70, p=0.000) higher than AR,  but there was no significant (AR: 
p=0.06, SC: p=0.30) difference between selection diet and regular diet (Table 5).  The 
slowest growth rate (15.82 ? 0.54 mg/day) was observed when AR larvae were exposed 
to selection diet. After seven days, when both AR and SC were transferred to or 
continued on regular diet, growth rates were significantly different (F
3,471
=12.34, 
p=0.000).  During this time, the growth rate in SC did not differ significantly (p=0.684) 
128 
 
based on their previous exposure.  However, initial exposure influenced the growth rate 
of AR larvae significantly (p=0.013).  The slowest growth rate (28.89 ? 0.62 mg/day) 
after seven days was observed in AR when they were initially exposed to regular diet. 
The difference in growth rate before and after seven days was significantly 
different (F
3,471
=3.84, p=0.010), and the highest difference (15.44 ? 0.77 mg/day) was 
observed when AR from selection diet was shifted to regular diet (Table 5).  Considering 
the change in growth rate on regular diet as 100% when SC was moved from selection to 
regular diet, growth rate increased by 107.35%.  However, in a similar comparison, the 
growth rate increase in AR was 131.18%. 
Fecundity and Fertility: Fertility and fecundity in AR on all types of diet could not be 
determined due to insufficient number of adults (result of high pupal mortality).  In SC, 
the fecundity and fertility were not influenced by the initial seven days exposure to diet 
of different strengths (Table 4).  
Fitness values after crossing AR with SC1: Larval mortality (F
1,5
= 11.148, p=0.029), 
pupal weight (F
1,151
= 15.426, p=0.000) and percentage malformed adults (F
1,5
= 53.646, 
p=0.002) differed significantly between AR1 and SC1 (Table 6).  However no significant 
differences were observed in larval weight after seven days (F
1,173
= 1.599, p=0.208), and 
larval (F
1,151
= 0.003, p=0.957) and pupal periods (F
1,104
= 0.229, p=0.633).  
Stability of resistance: Stability of resistance were conducted after only one generation 
of rearing on regular diet due to extremely high (range=40-80%) pupal mortality leading 
to the colony crashing.  After removing AR from Cry1Ac selection (referred as AR-Unsel 
in Fig. 4) for one generation, mean (? SE) percent survivors (10.2 ? 1.7) was reduced 
129 
 
significantly (t
1
=-7.78, p=0.016) compared to percent survivors (35.4 ? 1.54) when AR 
was under continuous selection (referred as AR-Sel in Fig. 4).   
Discussion 
In the present study, heritability (h
2
) of resistance, stability of resistance and fitness were 
assessed in a laboratory selected Cry1Ac-resistant strain of H. zea (AR).  Heritability (h
2
) 
values initially increased and then decreased over generations, indicating the increase in 
the genetic homogeneity of the population and hence, resistance factor.  At h
2
= 0.123, AR 
could develop 10-fold resistance to Cry1Ac in 10 generations at 30% selection pressure, 
which is less than the number of generations predicted for tobacco budworm, Heliothis 
virescens (Tabashnik 1992); possible reasons for quicker resistance evolution are 
discussed in Anilkumar et al. (2008).  
Fitness costs and the degree of dominance of fitness costs related to resistance 
determine the rate of resistance development (Carri?re et al. 1994).  In most studies, 
fitness costs were usually measured in the absence of the selection agent, presumably to 
approximate how long resistance would remain in the absence of field selection (Liu et 
al. 1999, Carri?re et al. 2001a, 2001b; Bird and Akhurst 2004, 2005).  This present study 
shows that under continuous selection AR had significantly higher pupal mortality, a 
male biased sex ratio, and decreased mating ability of moths compared to SC.  Increased 
pupal mortality for H. zea was also reported when larvae originated from Bt-corn (Storer 
et al. 2001) and Bt-cotton (Jackson et al. 2004a) compared to their non-Bt counterparts.  
The sex ratio in SC (0.47 ? 0.01) was similar to five batches of larvae (0.48 ? 0.01) 
collected from non-Bt field corn (during 2006 and 2007) (KJA and WJM, unpublished 
130 
 
data).  Further, the sex ratio of AR (0.51 ? 0.01) was similar to 167 larvae collected from 
a Bt-sweet corn field in 2006 (0.51) that were shown to be highly resistant to Cry1Ac 
toxin in the F
1
 generation (KJA and WJM, unpublished data).  Therefore, the male biased 
sex ratio in AR may be a result of resistance selection, suggesting higher susceptibility of 
females (De Lame et al. 2001, Shearer and Usmani 2001).  
The magnitude of mating costs
 
is expected to be influenced by factors such as 
mating
 
history, life span, current and past population sizes (bottlenecks,
 
founder effects), 
environmental conditions, and possibly interactions between these factors (Bird and 
Akhurst 2004).  SC is the parental population of AR (Anilkumar et al. 2008), both 
colonies were reared in parallel; genetic inbreeding independent from Cry1Ac selection 
seems an unlikely cause.  Even under conditions where there were significantly fewer 
adult AR compared to SC in a particular generation, the reduction in mating success for 
AR may not be linked to genetic inbreeding; AR in two generations (Aug-06 and Apr-07) 
had fewer (61 [30?:31?] and 58 [32?:26?]) adults but had increased (47 and 25% 
increase over previous generations) mating success past this potential bottleneck.  Percent 
mating for SC was similar to moths collected from light traps (Hendricks et al. 1970) and 
lower compared to collections made from sweep net and/or blacklight traps (Latheef et al. 
1991).  Further, mating increased in AR1 F
1
 adults, but was still significantly different 
from SC1. Additionally, AR1 F
2
 adults had reduced mating compared to their parents and 
the mating success was similar to AR before being crossed to SC1.  Therefore, reduced 
mating in AR may be due to Cry1Ac resistance and not necessarily inbreeding.  Further, 
reciprocal crosses indicate significant mating costs in males as against females.  Reduced 
131 
 
mating, mainly because of mating problems in males was also observed in Bt-resistant 
(selected using Dipel 2X) P. xylostella moths (Groeters et al. 1993).   
Fitness costs associated with resistance in AR have been demonstrated in many 
life history traits when reared on untreated diet.  Insects adopt different feeding strategies 
depending on the nutritional quality of the diet or host plants (Woods 1999).  Here, AR 
larvae exposed to selection diet had increased growth rate when shifted to regular diet, 
and with an additional day they achieved pupal weights similar to SC.  The increase in 
growth rate suggests increased feeding and/or higher assimilation rate, both of which may 
be due to an increased titer of digestive enzymes (Woods 1999).  Interestingly, AR 
produced a higher percentage of normal adults when exposed to toxin in selection 
experiments than when reared on untreated diet.  This may be due to 1) in the absence of 
selection, average fitness of individuals may decline due to the accumulation of 
deleterious mutations (Lynch et al. 1999),   2) elimination of higher percentage of insects 
with lower fitness (WJM, unpublished data), 3) AR has been selected with Cry1Ac toxin 
for 26 generations on selection diet containing 20% more water and therefore 20% less 
nutrients; AR have adapted to these conditions, as would be expected for a highly 
polyphagous insect (Woods 1999), and 4) exposure to Cry1Ac toxin affects the 
physiology of the insects such that they obtain higher fitness values from the increased 
nutrition of Bt (Sayyed et al. 2003); or other factors.  In the confused flour beetle, 
Tribolium confusum, reduced fitness was observed in a selection-free population 
compared to population with more intense selection (Lomnicki and Jasienski 2000).  
132 
 
AR required 27 days for adult eclosion on regular diet, compared to 25 days for 
SC.  This resulted in developmental asynchrony (Liu et al. 1999, Bird and Akhurst 2004, 
2005) as has been observed in other insects, and may lead to assortative mating (Liu et al. 
1999) thereby accelerating the rate of resistance evolution.  This should not be relative to 
H. zea, because peak mating occurs on the 4
th
 night after emergence (KJA, unpublished 
data, Shorey et al. 1968).  Assortative mating fitness differences will favor restoration of 
susceptibility in the absence of insecticide treatments (Groeters et al. 1993).   Caprio 
(2001), using a spatially descriptive model, found that non-random mating along with 
non-random oviposition could significantly delay resistance evolution.  
Long term rearing of insects in the laboratory results in reduced fitness mainly 
because of the founder effect and/or inbreeding (Roush and Daly, 1990). Therefore, AR 
was crossed to SC1 to ascertain whether observed reduction in fitness was linked to 
resistance (Bird and Akhurst 2004) and to save AR from extinction.  Even after one 
generation of crossing with SC1, AR1 had increased fitness costs while feeding on 
Cry1Ac-treated and untreated diet.  These observations strongly suggest that they may be 
linked to Cry1Ac-resistance as reported in Cry1Ac-resistant H. armigera after four 
crosses with a susceptible strain (Bird and Akhurst 2004).  Although AR1 appears similar 
to AR in terms of survivorship at 500 ?g Cry1Ac toxin/g diet, the RR for AR1 is lower 
than for AR because of increased tolerance of SC1 (LC
50
=31.25 ?g/g diet) compared to 
SC (LC
50
=9-15?g/g diet, Anilkumar et al. 2008).  This increased tolerance to Cry1Ac in 
SC1 may also come with fitness costs that were reflected in larval weight, larval and 
133 
 
pupal period not differing between AR1 and SC1; these fitness costs may not be linked to 
Cry1Ac-resistance.   
Resistance in AR was not stable; after one generation of rearing on regular diet 
AR lost a significant amount of resistance. Similar unstable resistance (from >500-fold to 
>74-fold) was also reported in Cry1C resistant Spodoptera littoralis (Muller-Cohn et al. 
1996).  However, Bt resistance was stable in Spodoptera exigua (Moar et al. 1995) and 
Plodia interpunctella (343-R) (McGaughey and Beeman 1988).  Both stable and unstable 
Bt resistance was observed in P. xylostella (Ferre and Rie 2002).  Unstable Cry1Ac 
resistance in AR may help in understanding observed reductions in the LC
50
 values of 
field collected populations which had elevated LC
50
 values in F
1,
 but declined rapidly 
during laboratory colonization (R. Luttrell, Univ. of Arkansas, personal communication).  
The reduction in resistance may be linked to fitness costs and/or accumulation of 
deleterious mutations (Lynch et al. 1999). 
Contrary to the initial expectations of rapid evolution of H. zea resistance to Bt-
cotton (Harris 1991, Roush 1997), there are no reports of field control failure(s) after 
more than a decade of Bollgard
?
 and Bt corn use (Ali et al. 2006, Moar and Anilkumar 
2007).  This lack of observed field-evolved resistance occurred despite widespread use of 
Bollgard
?
 and Bt corn during this period.  There are a number of mitigating factors which 
might have contributed to the delay of this pest developing resistance to Bollgard
?
; 1) the 
?high dose plus structured refuge?, 2) use of pyrethroid insecticide(s) to control 
bollworms during high infestations Bt-cotton (Anilkumar et al. 2008), 3) substantial 
temporal and spatial bollworm production from non-cotton crop hosts (Gustafson et al. 
134 
 
2006), and 4)  fitness costs associated with elevated Cry1Ac resistance or tolerance as 
shown in these studies and others.  The latter has likely played the most important role in 
delaying resistance development in bollworms. Indeed, Gustafson et al. (2006) 
incorporated assumed values (none, low and moderate) of fitness costs associated with 
either recessive or additive inheritance for resistance in modeling the effect of non-Bt 
crops as effective refuges for IRM.  For the Mississippi region, this model predicted a 
delay in resistance for 6-10, 7-14 and >30 yr with none, low and moderate fitness costs, 
respectively.  We believe that this model has been validated and may indicate even 
greater delays in resistance development if results from this study (moderate to high 
fitness costs) are incorporated in their model, assuming laboratory generated results are 
applicable to the field.   Recently, Tabashnik et al. (2008) reported field-evolved Cry1Ac-
resistance in H. zea based on laboratory assays of different strains collected from the field 
before (Luttrell et al. 1999) and after (Ali et al. 2006) commercial cultivation of Bt 
cotton. However, the conclusions of Tabashnik et al. (2008) are directly contradicted by 
the lack of observed changes in Bt cotton efficacy against H. zea and the lack of 
confirmed Bt resistant H. zea populations in the EPA-mandated Bt resistance monitoring 
program. We believe that the data presented in this present manuscript help to explain 
why field-evolved resistance has not yet occurred in this pest. 
 
Acknowledgements 
Authors thank N. Adams, Monsanto Co., Union City, TN for providing H. zea; E. van 
Santen, Dept. of Agronomy and Soils, and A. Appel, Dept of Entomology, Auburn 
135 
 
University for providing statistical guidance; and G. Head and S. Sivasupramaniam, 
Monsanto Co., St. Louis, MO, and J. Chandrashekhar for review of this manuscript.  This 
research was supported by the U.S. Department of Agriculture, and Cotton Incorporated. 
 
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142 
 
Table 1. Heritability (h
2
) and resistance risk assessment for resistance to Cry1Ac in 
H. zea   
N
a
 LC
50
 values RR
b
 R
c
 Slope S
d
 h
2e
 G
f
 
Initial
g
 Final
h
 Initial Final
4 8.89 107.64 12.11 0.271 1.71 1.42 0.860 0.315 3.69 
7 8.94 321.22 35.93 0.222 1.93 1.89 0.554 0.401 4.50 
11 11.82 1,450.00 122.7 0.190 1.76 1.76 0.742 0.256 5.27 
19 15.00 1,390.00 92.67 0.104 2.31 1.39 0.840 0.123 9.66 
LC
50
, resistance ratio and slope values are from Anilkumar et al. 2008 
a
N: Number of generations of continuous selection using Bt Cry1Ac treated diet; 
b
RR: Resistance ratio; 
c
R: response to selection; 
d
S: Selection differential;  
e
h
2
: heritability; 
f
G: Resistance risk = Number of generations required for 10-fold 
increase in resistance; 
g
Initial: LC
50
 for unselected parental strain; 
h
Final: LC
50
 for 
resistant strain measured after number of generations of selection  
 
 
143 
 
Table 2. Reproductive propensity (mean ? SE) of Cry1Ac-resistant (AR) and 
susceptible (SC) strains of H. zea during selection and rearing, respectively
a
. 
Strains N
b
 % mating % multiple 
mating 
# of Spermatophore 
/male 
AR 2066 40.27 ? 5.18 16.76 ? 3.40 0.58 ? 0.08 
SC 1820 71.85 ? 3.53 26.01 ? 2.34 1.23 ? 0.10 
t-Test results  t
13,1
= -6.468, 
p=0.000 
t
13,1
= -2.521, 
p=0.026 
t
13,1
=-5.628, 
p=0.000 
a
Results are from 14 generations of observations; 
b
N: Number of moths 
Superscript letters after means within a column indicate significant differences at p<0.05 
level by Tukey?s test 
 
 
144 
 
Table 3. The reproductive (mean ? SE) success in a Cry1Ac-resistant (AR), 
susceptible (SC) and their reciprocal crosses
a
  
Strain/cross N
b
 % mating % multiple 
mating 
Spermatophore 
/male 
AR 960 29.48 ? 2.98
a
 12.04 ? 1.93 0.41 ? 0.13
a
 
SC 815 66.29 ?  3.65
b
 21.60 ? 4.43 1.01 ? 0.39
b
 
AR ? X SC ? 140 58.48 ? 3.98
b
 20.19 ? 5.52 1.04 ? 0.32
b
 
SC ? X AR ? 143 40.24 ? 7.38
a
 16.10 ? 7.25 0.53 ? 0.30
ab
 
F-Test results  F
3,19
=14.29 
p=0.000 
F
3,19
=0.700 
p=0.566 
F
3,19
=5.772 
p=0.007 
a
Results are from five
 
generations of experiments, 
b
N: Number of moths  
Superscript letters after means within a column indicate significant differences at p<0.05 
level by Tukey?s test 
 
145 
 
Table 4. Fitness parameters (mean ? SE) for Cry1Ac-resistant (AR) and susceptible 
(SC) strains of H. zea
a
 
Life-history trait AR SC 
Regular diet Selection 
diet
b
 
Regular diet Selection 
diet 
Larval weight in 7D (mg) 109.48 ? 
3.22
b
 
91.04 ? 
3.89
a
  
144.58 ? 3.95
c
 131.99 ? 
4.48
c
  
Larval duration (d) 15.01 ? 
0.09
b
 
15.20 ? 
0.11
b
 
13.94 ? 0.11
a
 14.09 ? 
0.13
a
 
Larval mortality (%) 18.23 ? 
2.46
b
 
12.85 ? 
2.11
ab
 
5.04 ? 0.63
a
 10.07 ? 
3.91
ab
 
Pupal weight (mg) 347.70 ? 
4.22
a
 
355.41 ? 
3.37
ab
 
375.32 ? 
3.34
bc
 
368.4 ? 
3.67
bc
 
Pupal duration (d) 12.02 ? 
0.12
b
 
12.19 ? 
0.12
b
 
11.39 ? 0.1
a
 11.82 ? 0.1
b
 
Malformed adults (%) 74.38 ? 
13.46
b
 
71.10 ? 9.49
b
 
13.87 ? 1.88
a
 16.10 ? 
6.12
a
 
Number of eggs NA
c
 NA 653.74 ? 
51.58 
583.26 ? 
68.53 
Hatching (%) NA NA 85.29 ? 2.61 86.60 ? 3.12 
Superscript letters after means within a row indicate significant differences at p<0.05 
level by Tukey?s test 
a
Results are from 160 larvae;
 b
Selection diet (regular diet + 20% water [used for the 
purpose of adding Bt proteins into regular diet in resistance selection experiments]
 
c
NA: Not available, experiments were not continued due to higher percentage of 
malformed adults 
 
146 
 
Table 5. Growth rate (mean ? SE) for Cry1Ac-resistant (AR) and susceptible (SC) 
strains of H. zea on different strengths of diet
a
 
Strains Diet
b
 Growth rate (mg/day) 
During 7D After 7D Difference 
SC RD 21.09 ? 0.53
b
 32.93 ? 0.56
c
 11.83 ? 0.81
a
 
SD 20.39 ? 0.60
b
 33.09 ? 0.61
c
 12.70 ? 0.85
a
 
AR RD 17.12 ? 0.44
a
 28.89 ? 0.62
a
 11.77 ? 0.78
a
 
SD 15.82 ? 0.54
a
 31.07 ? 0.51
b
 15.44 ? 0.77
b
 
F-test results F
3,471
=22.70, 
p=0.000 
F
3,471
=12.34, 
p=0.000 
F
3,471
=3.84, 
p=0.010 
a
Results
 
from 160 larvae: 
b
RD: Regular diet, SD: Selection diet (RD + 20% water [used 
for the purpose of adding Bt proteins into regular diet]) 
Superscript letters after means within a column indicate significant differences at the 
p<0.05 level by Fisher?s least significant differences 
147 
 
Table 6.  Fitness parameters (mean ? SE) for Cry1Ac-resistant (AR1) and 
susceptible (SC1) strains of H. zea after crossing AR with SC1
a
 
 
Life-history trait AR1 SC1 
Larval weight in 7D (mg) 95.91 ? 6.38 88.99 ? 6.43 
Larval duration (d) 15.14 ? 0.17 15.24 ? 0.18 
Larval mortality (%) 22.70 ? 3.55
b
 9.69 ? 1.62
a
 
Pupal weight (mg) 359.06 ? 7.12
a
  391.72 ? 5.11
b
 
Pupal duration (d) 11.59 ? 0.20 11.72 ? 0.14 
Malformed adults (%) 60.81 ? 6.49
b
 10.77 ? 2.14
a
 
a
Results are from 96 larvae 
Superscript letters after means within life history traits indicate significant differences at 
the p<0.05 level by Tukey?s test 
148 
 
Fig 1. Pupal sex ratio of AR and SC strains over time with selection and rearing, 
respectively 
Months
Ja
n
-
0
6
F
e
b
-
0
6
A
p
r
-
0
6
Ju
n
-
0
6
J
u
l
-
0
6
S
e
p
-
0
6
O
ct
-
0
6
N
o
v
-
0
6
D
e
c-
0
6
F
e
b
-
0
7
M
a
r
-
0
7
A
p
r
-
0
7
M
a
y
-
0
7
Ju
n
-
0
7
Ju
l
-
0
7
N
o
v
-
0
7
D
e
c-
0
7
P
r
opor
t
i
on of
 m
a
l
e
s
0.40
0.42
0.44
0.46
0.48
0.50
0.52
0.54
0.56
0.58
AR 
SC 
AR crossed with SC1
 
149 
 
Fig 2. Mating success in AR and SC over time with selection and rearing, respectively 
 
Months
D
e
c
 
0
5
A
p
r
 
0
6
J
u
n
 0
6
A
u
g
 0
6
S
e
p
 0
6
O
c
t 
0
6
D
e
c
 0
6
J
a
n
 0
7
F
e
b
 
0
7
M
a
r
 
0
7
A
p
r
 
0
7
M
a
y 
0
7
J
u
n
e
 0
7
J
u
l
 
0
7
N
o
v
-
0
7
D
e
c
-
0
7
P
e
r
c
ent
age m
a
t
i
ng
0
20
40
60
80
100
 AR 
SC 
AR crossed with SC1 strain
150 
 
Fig 3. Growth rate differences in AR and SC, when larvae were reared on regular diet 
after exposing to regular diet and selection diet (20% diluted regular diet) for initial seven 
days. 
 
Strains and diet treatments
SC-RD SC-SD AR-RD AR-SD
G
r
o
w
th
 ra
te
 (m
g
/
d
a
y
)
0
5
10
15
20
25
30
35
7D 
After 7D 
Difference 
 
 
151 
 
Fig 4. Percent survivors of Cry1Ac-resistant H. zea at 500 ?g/g Cry1Ac toxin when 
selected continuously at 500 ?g/g Cry1Ac toxin compared to when removed from 
selection for one generation.  
AR-Sel AR-Unsel
%
 
S
u
r
v
i
v
o
r
s
 at
 50
0 
?
gC
r
y
1A
c/
m
g
0
10
20
30
40
 
152 
 
CHAPTER 4: 
SYNERGISTIC INTERACTIONS BETWEEN CRY1AC AND GOSSYPOL LIMIT 
SURVIVAL OF CRY1AC-RESISTANT HELICOVERPA ZEA (LEPIDOPTERA: 
NOCTUIDAE) ON BT COTTON 
 
ABSTRACT Results of laboratory experiments with field-cultivated cotton squares 
demonstrate that Cry1Ac-resistant Helicoverpa zea (Boddie) cannot complete larval 
development on Bt cotton, despite being more than 150-fold resistant to Cry1Ac.  Diet 
incorporation bioassays were conducted with Cry1Ac (15 ?g/g), gossypol (0.15%), their 
2, & 4-fold dilutions; and 4% corn and cotton powders in the presence and absence of 
Cry1Ac (15 ?g/g) to help determine the contribution of these compounds to the results 
observed using Bt cotton.  Cry1Ac toxin (15 ?g/g) was significantly more lethal to 
susceptible compared to resistant strain however no differential susceptibility was 
observed in strains for 0.15% of gossypol.  Combinations of Cry1Ac and gossypol, 4% 
cotton or corn powders were synergistic against resistant, but not to susceptible strain.   
Gossypol concentration in individual larvae showed no significant differences between 
strains, or between gossypol alone and gossypol plus Cry1Ac. These results may help 
understand the inability of resistant strain to complete development on Bt cotton, and 
therefore may help explain the absence of field-evolved resistance to Bt cotton by H. zea. 
 
153 
 
Introduction 
Bollworm, Helicoverpa zea (Boddie) has a naturally high tolerance to Bacillus 
thuringiensis (Bt) Cry1Ac compared to other target pests of Bt cotton in the US such as 
tobacco budworm Heliothis virescens (F.) and pink bollworm Pectinophora gossypiella 
(Saunders) (MacIntosh et al. 1990b, Sivasupramaniam et al. 2008).  This relatively high 
tolerance coupled with preferential feeding on tissues expressing lower levels of Bt 
protein (Brickle et al. 2001) and toxin attenuation due to abiotic stress and plant 
phenology can result in complete larval development on Cry1Ac cotton, especially under 
high insect populations (Jackson et al. 2004a).  Additionally, H. zea is also exposed to Bt 
Cry1Ab in Cry1Ab-expressing corn, which is similar in structure and function to Cry1Ac 
(Crickmore et al. 1998).  It has been postulated that this high selection pressure increases 
the likelihood of resistance evolution (Gould 1998) and it is therefore not surprising that 
models predicted resistance development within 3-7 years (Harris 1991, Roush 1997). 
Furthermore, the capacity for H. zea to develop resistance to Cry1Ac has been 
demonstrated in laboratory-selected strains (Luttrell et al. 1999, Jackson et al. 2004b, 
Anilkumar et al. 2008a). However, contrary to these results and predictions, field-evolved 
resistance has not occurred with H. zea even after 12 years of commercial use of Bt 
cotton in the USA (Ali et al. 2008). 
Although numerous Cry1Ac-resistant strains of H. virescens (Gould et al. 1992, 
1995), P. gossypiella (Liu et al. 1999, Tabashnik et al. 2000), and Helicoverpa armigera 
(Hubner) (Akhurst et al 2003), have been developed in the laboratory, very few strains 
have been able to pupate and produce fertile adults on Bt cotton (Tabashnik et al. 2003, 
154 
 
Bird and Akhurst 2004, 2005).  Of those Lepidopteran strains that could develop to fertile 
adults on Bt cotton, there is little correlation between the relative susceptibility of these 
strains to Cry1Ac, the level of Cry1Ac resistance in these resistant strains, and the 
relative survivorship of these resistant strains on Bt cotton (Liu et al. 1999, 2001a, 2001b, 
Akhurst et al. 2003, Tabashnik et al. 2003, Bird and Akhurst et al. 2004, 2005).  Possible 
explanations for this low correlation include interactions of Bt proteins with secondary 
plant metabolites (Carri?re et al. 2004), use of a form of Bt protein for resistance 
selection not exclusively found in Bt cotton (Liu et al. 1999, 2001a, 2001b, Tabashnik et 
al. 2000, Akhurst et al. 2003, Henneberry and Jech, 2007, Anilkumar et al. 2008a), 
increased consumption of plant tissues compared to consumption of artificial diet 
(Woods, 1999), loss of genes or a reduction in expression of compounds necessary to 
survive on cotton during laboratory rearing and Bt resistance selection, or fitness costs 
associated with Bt resistance (Tabashnik et al. 2003).  
Anilkumar et al. (2008a) reported a population of H. zea that was selected for 
stable and moderately high levels of resistance to the Cry1Ac toxin in the laboratory. 
Because H. zea is the most tolerant to Cry1Ac, and arguably the most polyphagous of all 
target lepidopteran pests of Bt cotton expressing Cry1Ac in the US, research was 
conducted to determine the survivorship of Cry1Ac-resistant and susceptible H. zea on 
field-cultivated Bt and non-Bt (NBt) cotton squares.  Further, this study also explored the 
interaction of Cry1Ac with gossypol, cotton powder and corn powder in artificial diet to 
help explain possible reasons for the higher than expected mortality observed for 
Cry1Ac-resistant H. zea in Bt cotton. 
155 
 
Materials and Methods 
Insect strains: A laboratory susceptible colony of H. zea (SC) was established in 
September 2004 from a laboratory colony from Monsanto (Union City, TN). A resistant 
strain (AR) was the product of selecting SC for resistance by exposing individual 
neonates to an artificial diet containing 500 ?g Bt Cry1Ac toxin/gram diet for 25 
generations (Anilkumar et al. 2008a).  Seven days after exposure to the Cry1Ac toxin, 
surviving molted larvae were transferred to an untreated diet and reared until pupation 
(Ali et al. 2006, Anilkumar et al. 2008a, Sivasupramaniam et al. 2008).  Resistance was 
assessed at selected generations (Anilkumar et al. 2008a) and AR was >150-fold resistant 
(based on artificial diet bioassays) at the time when survivorship bioassays on Bt and 
non-Bt cotton squares were conducted.   
Cry1Ac interactions with gossypol, cotton/corn powder: In order to avoid complete loss 
of AR due to fitness costs associated with Cry1Ac resistance selection and rearing in the 
laboratory for 26 generations (Anilkumar et al. 2008b), AR was crossed with a new 
Monsanto susceptible strain (from Union City, TN), SC1, resulting in AR1.  SC1 had 
higher LC
50
 (31.25?g Cry1Ac toxin/g of diet) values compared to SC (8.89 - 15 ?g 
Cry1Ac toxin /g of diet; Anilkumar et al. 2008a). Although both reciprocal crosses were 
attempted, only AR[?] X SC1[?] yielded a viable F
1
 population due to mating costs 
associated with AR males (Anilkumar et al. 2008b). Because Cry1Ac resistance in H. zea 
is inherited as a co-dominant character (Burd et al., 2003, Anilkumar et al. 2008b), high 
levels of resistance (resistance ratio {RR}>50-fold compared SC1) was observed in F
1
 
(data not shown) confirming that AR1 was resistant to Cry1Ac toxin. AR1 was selected 
156 
 
at the regular selection concentration of Cry1Ac (500 ?g Cry1Ac toxin/g of diet) for two 
generations (Anilkumar et al. 2008a). 
Cry1Ac toxin: An E. coli strain expressing Cry1Ac protoxin from B. thuringiensis 
subsp. kurstaki strain HD-1 (provided by L. Masson, Biotechnology Research Institute, 
National Research Council, Montreal, Canada) was cultured, and the activated toxin 
prepared as indicated elsewhere (Moar et al. 1994, Pusztai-Carey et al. 1994).  
Lyophilized leaf tissue powder studies: Lyophilized corn (LH198/LH172) and cotton 
(C312) leaf tissue powders were supplied by Monsanto.  
Plant Studies 
Cotton plants: Bt-cotton (DPL555) and the near isogenic non-Bt cotton (NBt) (DPL491) 
were planted at the Prattville Agricultural Research Unit, Alabama Agricultural Research 
Station, Prattville, AL. Planting dates for Bt- and NBt-cotton were April 23
rd
 and 24
th
, 
2007, respectively. Cotton plants were cultivated as per typical practices. NBt-cotton 
plants were treated with Imidacloprid at 1 oz/A on both July 13, and July 24, 2007 for 
aphid control and tissues were used five days after treatment for bioassays. Cotton plants 
were >85 days old when squares were harvested.  Pin-head to midpoint-stage squares (7-
14 D old) were collected, transported to the laboratory, and stored at 4-7 
0
C until needed, 
up to a maximum of 13 d. 
Cry1Ac protein quantification: Beginning on the day of field collection, and on days 
when cotton squares were removed from refrigeration and used in bioassays, a random 
sample of 10 Bt squares was placed at -80 
0
C.  After all bioassays were completed, all -80 
0
C samples were shipped to Monsanto (St. Louis, MO) for Cry1Ac protein quantification.  
157 
 
Square tissues were lyophilized, and Cry1Ac expression was determined using ELISA 
and compared against a positive Bt cotton standard as described in Greenplate (1999) and 
Sivasupramaniam et al. (2008).  Three replicate assays were conducted for each sample. 
Survival and development on squares: Individual neonates from both strains were 
placed on the outside of the square bracts on one square in a petri dish (35x10mm). Moist 
cotton was placed below squares to reduce desiccation, and squares were changed every 
three days (replacing larvae on bracts) until experiments were concluded.  For both Bt 
and NBt-cotton tests, larvae and squares were transferred to 30 ml cups containing three-
five squares when larvae reached 4
th
 instar, and rearing was continued until pupation. 
Observations on larval mortality and stadia were recorded beginning on the fourth day 
and subsequently at three day intervals. Larval weights were recorded after seven days 
and tests were continued until survivors reached pupation.  Data on larval mortality, 
larval weight after seven days, and larval duration were recorded as discussed above. 
Thirty larvae from each strain were tested in each replication and experiments were 
repeated three times. 
Gossypol studies 
Effect of Cry1Ac and gossypol: Growth and development of AR1 and SC1 were 
evaluated in diet containing 3.75, 7.5, 15 ?g Cry1Ac/g diet, or 0.0375, 0.075, 0.15% 
gossypol (95% in acetic acid crystals, Sigma, St. Louis, MO), and their 1:1 combination 
(at respective dilutions from the maximum concentration used); and were compared to an 
untreated control. Because gossypol was dissolved in 1.0 % Dimethylsulfoxide (DMSO), 
all treatments contained a final concentration of 1.0% DMSO. Gossypol and/or Cry1Ac 
158 
 
toxin were added when diet temperature was <60 
0
C, mixed thoroughly and poured into 
128 well bioassay trays (CD-International, Pitman, NJ) at one gram per well.  0 - 16 h old 
neonates were transferred individually into each well and covered with ventilated covers. 
Bioassay trays were incubated at 27 ? 1 
0
C and RH 50% and a photoperiod of 14:10 
(L:D) h.  Larval mortality, instar and weight were recorded after seven days.  Thirty two 
larvae were tested for each treatment and the experiment was replicated three times. 
Gossypol quantification:  Five larvae from each replication that survived after 7 days in 
bioassays above containing gossypol (gossypol alone, gossypol plus Cry1Ac) were 
weighed and placed individually in 30ml plastic cups containing no diet for 10-12 hours 
to allow for purging of gut contents.  Insects were transferred individually to a 
microcentrifuge tube and frozen at -80 
0
C. Gossypol content per insect was determined 
using method by Orth et al. (2007) 
Lyophilized leaf tissue powder studies 
Effect of Cry1Ac and corn/cotton powder: The performances of AR1 and SC1 on 4% 
cotton powder, 15 ?g Cry1Ac/g diet, and their 1:1 combinations were studied in two 
generations (six replications of 32 larvae/treatment). In one generation (three replicates of 
16 larvae/treatment) both strains were evaluated for their susceptibility to 4% corn 
powder and its interaction with Cry1Ac (15 ?g/g diet), and compared to the untreated 
control.  The experimental procedure was similar as explained above except for the 
absence of DMSO.  Further, larvae feeding on cotton powder alone and cotton powder 
plus Cry1Ac were assayed for gossypol content following the procedure discussed above. 
 
159 
 
Data analysis: 
Plant Studies: Age of the plant at sampling was considered a fixed classification effect 
and duration of storage as a fixed effects covariate nested with age to analyze the effect 
of storage on Cry1Ac stability. The sole random effect in the model was replicate(age) 
and Proc Mixed predicted a separate intercept for each age class with the no intercept 
option (SAS Institute 2003).  
Larval mortality was modeled as logistic regression with SAS Proc NLmixed 
using the binomial distribution function. The degrees of freedom for t-tests and 
confidence intervals were calculated as the number of group means minus the number of 
fitted parameters (Schabenberger and Pierce 2004).  We used the CONTRAST statement 
to evaluate the statistical significance of toxin differences within strains and ESTIMATE 
to calculate LT
50, 
and LT
75
 values plus associated 95% confidence intervals as well as the 
contrasts between strains. 
The effect of diet on larval development (instars 3 and 4) was modeled with SAS 
Proc GLIMMIX with a binomial distribution function and the logit link function.  Strain, 
treatment and their interactions were fixed effects and replicate the sole random effect.   
Gossypol studies: For larval weight data, replicate x strain interaction means in response 
to toxin rate were modeled with SAS Proc NLmixed using an exponential decay model 
with a lower asymptotic limit and a normal distribution function.  To improve the ability 
to detect otherwise small differences between strains and treatments, failure to molt to 
third instars was considered as mortality and analyzed as discussed above.  Due to the 
relatively small effect of gossypol alone on mortality, lethal concentrations for Cry1Ac 
160 
 
and gossypol mixtures were estimated in terms of concentration of Cry1Ac.  Effective 
concentrations (EC) were calculated similar to the calculation of lethal concentrations.  
EC
50
 is defined as the effective concentration of toxin which provides 50% weight 
reduction in a test population (Jalali et al. 2004).   
The distribution of gossypol concentration per larva was right skewed; therefore, 
the data was analyzed using a generalized linear models framework utilizing SAS Proc 
GLIMMIX. The lognormal distribution function resulted in a symmetrical distribution of 
residuals. Strain and toxin were fixed effects class variables and toxin rate was treated as 
a fixed effects covariate.  Differences among toxin x strain combinations were then 
predicted at toxin rates 375, 750, and 1,500 using the AT option of the LSmeans 
statement with the simulation adjustment to control the Type I error rate. 
Interactions of Cry1Ac and gossypol were evaluated as described by Salama et al. 
(1984).  Differences in observed mortality and theoretical mortality for the mixture of 
Cry1Ac and gossypol were analyzed using ?
2
 tests.  Interaction was considered 1) 
synergistic, if observed mortality was more than expected mortality coupled with 
significant chi square values, 2) additive, if observed mortality was more than expected 
mortality coupled with non significant chi square values, 3) antagonistic, if observed 
mortality was less than expected mortality coupled with significant chi square values. 
Lyophilized leaf tissue powder studies: Larval mortality was modeled with SAS Proc 
GLIMMIX with a normal distribution function.  The residual variance was modeled 
using the group option to account for heterogeneous variances among treatments.  Larval 
weight and gossypol concentration/mg larva were modeled using the same procedure but 
161 
 
with lognormal distribution function, which was necessary as residuals under the normal 
assumption were extremely right-skewed.  Treatment, strain and their interaction were 
treated as fixed effects. However random effects were different for each of data 
parameters.  For larval weight, generation, replicate (generation) and their interactions 
with fixed effects. For larval mortality, generation and replicate (generation) were 
considered to be random effects.  Least squares diet x strain interaction means were 
calculated.  The slicediff (for larval weight and mortality), pdiff (gossypol 
concentration/larvae) and simulation options were employed to assess differences among 
strains and diets while controlling the Type I error rate.  Analysis of synergism between 
Cry1Ac and cotton/corn powders were evaluated as explained above.  
Results 
Cry1Ac expression in squares: The concentration of Cry1Ac in Bt cotton squares 
quantified using ELISA were not significantly different from levels of Cry1Ac found in 
the positive control, DP50 Bollgard
? 
squares (Fig 1).  Cry1Ac expression was reduced 
significantly (F
5
=248.75, P < 0.0001) after 86 days (July 18) after planting; and storage 
at 4-7 
0
C did not affect the stability of the Cry1Ac for all samples except the July 18
th
 
samples (P = 0.0488). 
Survivorship of AR and SC on Bt-cotton and NBt-cotton: LT
50 
(lethal time for 50% 
mortality) values for AR (9.13 d) on Bt squares was significantly (P=0.0004) higher 
compared to SC (LT
50
 = 4.75 d).  Similarly, LT
75
 values were significantly (P<0.0001) 
higher for AR compared to SC (Table 1). Contrastingly, there were no significant 
differences in LT
50 
values for
 
either SC (23.18 d) or AR (22.33 d), larvae on NBt squares. 
162 
 
Larval weight after seven days was significantly different between Bt and NBt 
cotton tissues (P < 0.0001) but not between strains (P = 0.4056) and their interaction 
with tissues (P = 0.1248) (Table 2).  Tissue (P < 0.0001) and its interaction (P = 0.0278) 
with strain but not the strain alone (P = 0.0985) had a significant effect on the number of 
larvae reaching third instar.  The proportion of larvae reaching third instar differed 
significantly between AR and SC on Bt but not on non-Bt tissues and only AR reached 
fourth instar on Bt squares.   
Effect of Cry1Ac and gossypol:  LC
50 
(?g/g diet) values were significantly different 
between AR1 (17.56) and SC1 (7.07) for Cry1Ac. In the presence of gossypol, however, 
the LC
50
 for AR1 (10.04) was decreased substantially and not significantly different (P = 
0.05) from SC1 on Cry1Ac alone (Table 3).  AR1 (10.04) and SC1 (5.41) strains had 
significantly different LC
50
 values for the Cry1Ac and gossypol mixture.  EC
50
 (?g/g 
diet) values for both AR1 (1,172) and SC1 (1,204) were similar for gossypol but were 
significantly higher compared to Cry1Ac (AR1=0.99, SC1=0.97) and their mixtures 
(AR1=1.35, SC1=0.94) (Table 4). 
Gossypol concentration/larvae did not differ significantly between strains at any 
concentration (375, 750 and 1,500?g/g diet) of gossypol in the presence or absence of 
Cry1Ac (Fig. 3A).  However, irrespective of strains, larvae feeding on 0.15% gossypol in 
the presence of 15 ?g Cry1Ac/g of diet had significantly lower gossypol/mg body weight.    
Effect of Cry1Ac and cotton/corn powder: Strain (F
1
=49.25, P < 0.0001), treatment 
(F
5
=672.10, P < 0.0001), and the strain x treatment interaction (F
5
=44.27, P < 0.0001) 
had a significant effect on larval mortality as measured by failure to molt to third instar 
163 
 
(Table 5).  Significant differences between strains were observed in Cry1Ac (P < 
0.0001), corn powder (P = 0.0065) and their mixtures (P = 0.0002).  However, no 
significant differences were observed between strains in untreated diet (P = 0.9333), 4% 
cotton powder in the presence (P = 0.0506), or absence (0.2616) of Cry1Ac treatments.  
The mortality in mixtures of corn or cotton powders with Cry1Ac were significantly (P < 
0.0001) different in AR1 even though AR1 was not (P = 0.6680) differentially 
susceptible to either cotton or corn powder.  However, no such differences were observed 
in SC1. 
Gossypol concentration/larvae did not differ significantly between strains (F
1
=0.0, 
P = 0.9853) and its interaction (F
2
=0.70, P = 0.5010) with treatment.  However, larvae 
feeding on different treatments had significantly (F
2
=243.84, P < 0.0001) different levels 
of gossypol (Fig. 3B).   
Treatments (F
5
=244.01, P < 0.0001), strains (F
1
=17.14, P = 0.0005) and their 
interactions (F
5
=16.85, P < 0.0001) influenced larval weight significantly (Fig 4).  AR1 
and SC1 larval weights differed significantly in Cry1Ac (P < 0.0001), and its mixture 
with 4% corn powder (P = 0.0027) but not in other treatments.  
Synergistic interactions:  Considering failure to molt to third instar as mortality, 
significant synergistic interactions of Cry1Ac with both corn and cotton powders and 
gossypol were observed only for AR1 compared to SC1 (Table 6).  There was only an 
additive interaction at lower levels of Cry1Ac and gossypol in the diet.  Further, the level 
of synergism varied between corn and cotton powders with Cry1Ac.   
 
164 
 
Discussion 
ELISA results support the conclusion that >150-fold Cry1Ac-resistant H. zea (AR) would 
not be able to complete development on Bt cotton squares in commercial fields.  ELISA 
results are particularly important for this study as Prattville, AL, sustained one of its 
worst droughts (summer 2007) in history, documenting that Bt cotton can still produce an 
efficacious amount of Cry1Ac toxin even under drought conditions. Mean Cry1Ac 
expression was reduced by 45% from 86 to 124 d after planting which is similar to 
published results  by Greenplate (1999).  Refrigeration of harvested squares at 4-7 
0
C for 
up to 13 d did not result in a significant reduction in Cry1Ac levels. These results may 
help future investigations in which Bt cotton squares will need to be refrigerated for 
extended periods prior to use.  
Our results support the use of Cry1Ac toxin for resistance selection, and that this 
selection can confer partial resistance in H. zea to Bt cotton that expresses full-length 
protoxin although some to most may have been processed to active toxin by the time Bt 
protein is ingested (Anilkumar et al. 2008a, Gao et al. 2006, Li et al. 2007).  Evaluation 
of survivorship using squares was justified as young buds (pinhead square) and fruiting 
structures (squares, flowers, bracts and bolls) are the preferred oviposition and feeding 
sites for H. zea (Torres and Ruberson 2006).  Increased survivorship of AR on Bt cotton 
could be attributed to >150-fold level of resistance to Cry1Ac toxin developed in the 
laboratory (Anilkumar et al. 2008a) and also the increased feeding of AR larvae on bracts 
compared to SC (KJA and WJM, unpublished data), which express lower levels of toxin 
compared to other square tissues (Sivasupramaniam et al. 2008).  
165 
 
H. zea is >100-fold less susceptible to Cry1Ac than P. gossypiella (LC
50
=0.1 
?g/g) (Anilkumar et al. 2008a, Sivasupramaniam et al. 2008, Liu et al. 2001b). Therefore, 
AR would be expected to survive on Bollgard
?
, and at RR?s significantly less than those 
deemed necessary for a Cry1Ac-resistant insect such as P. gossypiella (>100-fold and 
>3,100) (Liu et al. 1999, 2001a, Tabashnik et al. 2003).  However, AR could not develop 
to pupation on Bt cotton, even with >150-fold resistance to Cry1Ac toxin (based on 
artificial diet bioassays).  Interestingly, Cry1Ac-resistant H. armigera have been shown 
to survive on Bt cotton and produce fertile adults (Bird and Akhurst 2004, 2005). 
Although H. zea (Anilkumar et al. 2008a, MacIntosh et al. 1990b) and H. armigera 
(Akhurst et al. 2003) have similar baseline susceptibility, there are many reports from 
Australia, China (0.09 ? 9.07 ?g/ml, Wu et al. 1999), India (0.01? 0.71 ?g/ml, Kranthi et 
al. 2001, Jalali et al. 2004) and Spain (3.5 ?g/ml, Avilla et al. 2005) showing variability 
in significant susceptibility of H. armigera to Cry1Ac.  These differences may be due to 
1) quantity of toxin expressed in the plant, 2) presence of Cry1Ac in both protoxin and 
toxin form in the plant (Anilkumar et al. 2008a), 3) interaction of toxin and other plant 
traits (Tabashnik et al. 2003, Carri?re et al. 2004), 4) larval age, 5) Bt resistance 
mechanism, 6) insect species and population variation and 7) variation in Bt cotton 
cultivar and growing conditions.   
H. armigera (Bird and Akhurst 2004, 2005) and P. gossypiella (Liu et al. 1999) 
could survive to pupation on Bt cotton when selected on artificial diets containing 
Cry1Ac protoxin or MVP II, respectively.  However, when P. gossypiella was selected 
for 42 generations using Bt cotton bolls (NuCOTN33B), larvae could not complete 
166 
 
development on Bt cotton and this strain did not have a reduction in cadherin binding 
(Henneberry and Jech 2007); similar to reports by Anilkumar et al. (2008a) for Cry1Ac 
toxin-selected H. zea.  Therefore, the Cry1Ac form and method of administration 
(artificial diet vs. plant material) may play an important role when determining RR?s 
needed for surviving on Bt cotton.  
There were no significant differences in AR and SC survivability and time 
required for pupation on NBt cotton.  However, Cry1Ac-resistant H. virescens 
(Tabashnik et al. 2003), H. armigera (Bird and Akhurst 2004, 2005) and P. gossypiella 
(Liu et al. 1999) had significantly slower larval development on NBt cotton and lower 
adult overwintering survival than susceptible individuals (Carri?re et al. 2001, Bird and 
Akhurst 2004). Additionally, Cry1Ac-resistant P. gossypiella had less tolerance to 
gossypol than susceptible individuals, resulting in fitness being decreased by >50% 
(Carri?re et al. 2001, 2004).  In our present study, however, there were no observable 
fitness costs compared to SC1 when gossypol alone was tested against AR1.  Further, 
gossypol concentration per larvae varied significantly between treatments (gossypol 
alone and 1:1 mixture of gossypol and Cry1Ac) but not between strains (AR1 and SC1) 
only at the highest concentrations tested. At the highest concentration of the 1:1 mixture, 
both strains recorded lower larval weight due to inhibition of feeding induced by pure 
gossypol (Meisner et al. 1976) and Cry1Ac (Whalon and Wingerd 2003) resulting in 
significantly less gossypol per body wt.  These results suggest that plant secondary 
metabolites other than gossypol, and/or nutritional factors, may adversely affect AR 
fitness (Tabashnik et al. 2003). 
167 
 
Although AR1 appears similar to AR in terms of survivorship at 500 ?g Cry1Ac 
toxin/g diet, the RR appears lower for AR1 because of the increased tolerance of SC1 
(LC
50
=31.25 ?g/g diet) compared to SC (LC
50
=9-15?g/g diet, Anilkumar et al. 2008a). 
Only LC
50 
but not EC
50
 values were different between AR1 and SC1 for Cry1Ac; which 
is due to 1) larval weight does not change significantly a day before and after molting, 2) 
EC
50
 values are estimated compared to larval weight in untreated diet treatment.  Cry1Ac 
toxin was tested at 15 ?g/g of diet, a concentration which is similar to the amount 
quantified from Bt cotton squares using ELISA. At this concentration 54% of AR1 could 
develop to pupation (data not shown), suggesting that AR should be able to develop to 
pupation on Bt cotton especially if larvae feed selectively on tissues expressing lower 
Cry1Ac concentrations compared to squares (Sivasupramaniam et al. 2008).  However, 
none of the AR reached pupation on Bt cotton.  Results from Cry1Ac interactions with 
gossypol, and cotton powder showed synergistic interactions to AR1 but not to SC1 when 
mortality was measured as failure to molt to third instar; mortality defined as dead larvae, 
or dead plus larvae in first instar, could not explain the differences observed between 
treatments.  Synergistic interactions of gossypol and Cry1Ac were observed only at the 
highest concentrations tested.  Furthermore, 4% cotton powder was synergistic to AR1 
although it contained only 6 ?g gossypol/g of diet (much less than the lowest 
concentration of gossypol tested that showed no differences in activity between SC1 and 
AR1).  In addition to gossypol, cotton plants produce many other insecticidal secondary 
metabolites such as heliocides H
1
 and H
2
, hemigossypolone, etc., (Hedin et al. 1991).  
Therefore, these compounds could potentially interact to reduce AR survivorship on NBt 
168 
 
cotton.  Additionally, 4% corn powder was also found to be interacting synergistically 
with Cry1Ac against AR1 but not to SC1.  Although, corn does not have gossypol it has 
many secondary metabolites such as zeatin, which is found to affect the signaling 
pathway (jasmonate and salicylate) in plants when damage occurs through insect 
herbivory resulting in the activation of cytochrome P450 production.   
Concentrations of gossypol and Cry1Ac used in this study were based on dry 
weights, which may not be representative of what an insect might consume while feeding 
on plants, as nearly 95% of total plant weight is water.  However, because the nutritional 
value of plant material is significantly lower than nutritionally-rich artificial diet, larvae 
consume 6-8-fold less artificial diet compared to feeding on plant material (Naeem et al. 
1992, Woods 1999). Therefore, we believe that the concentrations of gossypol and 
Cry1Ac used in this study are justifiable.   
This is the first report of plant compounds other than protein inhibitors 
synergizing the activity of Bt proteins, especially against Cry1Ac-resistant insects. To 
date most compounds that are synergistic with Bt Cry proteins are other Bacillus spp or 
Bt products such as spores or spore crystal mixtures (Tang et al. 1996, Liu et al. 1998, 
Moar et al. 1989, 1995), zwittermicin A (Broderick et al. 2000), ?-exotoxin (Moar et al. 
1986) and CytA (Wirth et al. 1997) and a peptide expressed in E. coli containing a 
corresponding Bt binding sequence (Chen et al. 2007). However, plant protease inhibitors 
and several chemical insecticides have also been reported to synergize Bt proteins, 
mixtures (Herfs et al. 1965, MacIntosh et al. 1990a).  Gossypol occurs naturally in an 
enantiomeric mixture of both (+)-gossypol and (-)-gossypol, and their ratio varies among 
169 
 
commercial cultivars.  Both of these forms reduced the survivorship of H. zea, and a 
racemic mixture of 1:1 had a synergistic effect at 0.16% (Stipanovic et al. 2006). The 
gossypol obtained from Sigma used in the current study was extracted from cotton seeds, 
and the ratio of enantiomeric forms of gossypol was not provided.  Therefore, further 
studies are warranted to quantify the ratios of enantiomers and to evaluate their 
interactions with Cry1Ac  
The synergistic interaction of gossypol, and cotton with Cry1Ac observed in AR1 
may help explain the inability of AR to survive and produce fertile adults on Bt cotton.  
Carri?re et al. (2004) suggested that increased susceptibility of Cry1Ac-resistant P. 
gossypiella to gossypol was linked to the cadherin mutation resistance mechanism (Morin 
et al. 2003, Carri?re et al. 2006).  However, AR has not been shown to have any 
differences in Cry1Ac binding (essentially eliminating a cadherin mutation as a potential 
resistance mechanism), and is speculated to have altered proteolysis as a resistance 
mechanism (Anilkumar et al. 2008a).  Additionally, AR1 was not differentially 
susceptible to gossypol alone compared to SC1. Therefore, future studies are warranted to 
determine how resistance mechanisms not associated with binding (or fitness costs 
involved with Cry1Ac selection) are affected by the presence of gossypol and other plant 
compounds.   
Predicting field-evolved Bt resistance based on laboratory studies has always been 
tenuous.   Field-evolved resistance has not occurred with H. zea even after 12 y of 
commercial use of Bt cotton in the USA (Ali et al. 2006, 2008, Moar and Anilkumar 
2007) even though laboratory experiments have shown that H. zea does have the 
170 
 
capability to become resistant to the Bt protein in Bt cotton (Cry1Ac) (Luttrell et al. 1999, 
Jackson et al. 2004b, Anilkumar et al. 2008a).  Results presented in this study help to 
illustrate that the actual hurdles that H. zea must overcome to become resistant to Bt 
cotton in the field are most likely quite complex and help to validate the absence of field-
evolved resistance in H. zea. 
Acknowledgements 
The authors thank Nancy Adams, Monsanto Co., Union City, TN for providing H. zea; 
Arthur Appel, Dept of Entomology, Auburn University for providing statistical guidance; 
and Marianne Carey for providing purified Cry1Ac toxin.  This research was partially 
supported by USDA, and Cotton Incorporated.  
 
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180 
 
Table 1. Lethal time (LT) to mortality for a Cry1Ac-resistant (AR) and a susceptible (SC) 
H. zea on Bt (DPL-555) and NBt (DPL-491) cotton squares.  The test was conducted in 
three replicates of 30 insects each; hence the df for P-values and confidence intervals was 
4. 
Squares Strain LT
50
(95% CI) 
(Days) 
LT
75
(95% CI) 
(Days) 
Slope ? SE 
Bt 
AR 9.13 (6.99 - 9.13) 13.41 (12.26 ? 14.57) 4.50 ? 0.36
SC 4.75 (3.94 ? 5.55) 7.80 (6.95 ? 8.65) 4.94 ? 0.51
NBt 
AR 22.33 (20.78 - 23.88) 31.33 (28.75 ? 33.92) 4.03 ? 0.36
SC 23.18 (21.56 ? 24.80) 32.30 (29.57 ? 35.03) 4.11 ? 0.37
 
181 
 
Table 2. Performance of a Cry1Ac-resistant (AR) and a susceptible (SC) strains on Bt 
(DPL-555) and NBt (DPL-491) cotton squares. The test was conducted in three replicates 
of 30 insects each.  
Square Strain Larval mass at 7d (mg) Proportions at the end of the experiment (31 d) 
3
rd
 instars 4
th
 instars 
Bt AR 0.93 (0.69 ? 1.25)
a
 0.19(0.07 - 0.42) 0.06 (0.02 ? 0.15) 
SC 0.66 (0.45 ? 0.96) 0.05 (0.02 - 0.16) 0.00 
NBt AR 4.11 (3.16 ? 5.35) 0.76 (0.51 ? 0.91) 0.63 (0.51 ? 0.74) 
SC 4.57 (3.51 ? 5.95) 0.81 (0.58 ? 0.93) 0.66 (0.53 ? 0.76) 
F-Test    
Strain F
1
=0.73 P = 0.4056 F
1
=3.49 P = 0.0985 F
1
=0.00 P = 0.9537 
Tissue F
1
=155.74  P < 0.0001 F
1
=108.0 P < 0.0001 F
1
=0.00 P = 0.9697 
Strain* Tissue F
1
=2.66  P = 0.1248 F
1
=7.20 P = 0.0278 F
1
=0.00 P = 0.9692 
a
values in the parenthesis are 95% confidence intervals
182 
 
Table 3. Molt inhibitory concentration (failure to molt to third instar) response of a 
Cry1Ac-resistant (AR1) and a susceptible (SC1) H. zea to Cry1Ac, and its 1:1 mixture 
with gossypol. The test was conducted in three replicates of 32 insects each. 
Treatments Strain LC
50 
(?g/g of diet)
a
 Slope  Intercept  
Cry1Ac AR1 17.56 (12.02 ? 23.10)
b
 0.10 (0.05 ? 0.15) -1.78 (-2.28 ?  -1.29) 
SC1 7.07 (5.99 ? 8.75) 0.29 (0.22 ? 0.35) -2.87 (-3.51 ?  -2.24) 
Cry1Ac + 
Gossypol 
AR1 10.04 (8.28 ? 11.27) 0.29 (0.22 ? 0.36) -2.02 (-2.54 ?  -1.50) 
SC1 5.41  (4.68 ? 6.14) 0.48 (0.36 ? 0.60) -2.60 (-3.29 ?  -1.91) 
 
a
LC
50
 values for mixture of Cry1Ac and gossypol are expressed concentrations of 
Cry1Ac; 
b
values in the parenthesis are
 
95% confidence intervals and are in logit scale. 
 
183 
 
Table 4.  Weight stunting concentration response of a Cry1Ac-resistant (AR1) and a 
susceptible (SC1) strain of H. zea to Cry1Ac, gossypol and their 1:1 mixtures. The test 
was conducted in three replicates of 32 insects each. 
Treatments Strains EC
50
 (?g/g of diet)
a
 EC
90 
(?g/g of diet)  
Gossypol AR1 1,171.81 (892.63 ? 1,450.98)
b
 2,109. 25 (1,606.73 ? 2,611.77) 
 SC1 1,204.01 (916.35 ? 1,491.66) 2,167.21 (1,649.43 ? 2,684.99) 
Cry1Ac 
AR1 0.99 (0.12-1.87) 3.30 (0.39 - 6.20) 
SC1 0.97  (0.31-1.63) 3.23 (1.04 - 5.42) 
Cry1Ac + 
Gossypol 
AR1 1.35  (0.52 - 2.18) 4.48 (1.72 - 7.25) 
SC1 0.94 (0.26 - 1.63) 3.14 (0.86 - 5.41) 
a
EC- Effective concentration (related to stunting - weight related). EC
50
, EC
90
 ? 
Concentration of Cry1Ac that would stunt the larvae such that they weighed 50 and 10% 
of that of larvae in the untreated control group; values for Cry1Ac + gossypol are 
expressed in concentrations of Cry1Ac; 
b
values in the parenthesis are
 
95% confidence 
intervals. 
 
 
184 
 
Table 5. Percent mortality of a Cry1Ac-resistant (AR1) and a susceptible (SC1) H. zea in 
4% cotton/corn powder in the presence and absence of 15 ?g Cry1Ac/g diet. 
 
Treatments N
a
 Failure to molt to 3
rd
  instar (mean ? SE) 
AR1 SC1 P value 
Untreated diet 192 11.59 ? 2.66
a
 11.98 ? 3.90
a
 0.9333 
Cry1Ac 200 20.59 ? 3.04
a
 86.98 ? 3.27
b
 < 0.0001 
Cotton powder 222 22.65 ? 3.99
a
 14.23 ? 5.94
a
 0.2616 
Cry1Ac + cotton powder 205 98.31 ? 0.69
c
 96.01 ? 1.09
b
 0.0506 
Corn powder 48 15.14 ? 1.81
a
 4.65 ? 2.37
a
 0.0002 
Cry1Ac + corn powder 48 53.21 ? 6.79
b
 95.83 ? 2.08
b
 0.0065 
Strains  F
1
=49.25,  P < 0.0001 
Treatment  F
5
=672.10,  P < 0.0001 
Strain * Treatment  F
5
=44.27,  P < 0.0001 
 
a
Number of insects tested;  means within a column followed by different superscript 
letters are significantly different at P = 0.05
185 
 
Table 6. Interactions of Cry1Ac with gossypol, cotton and corn powder as measured by 
failure to molt into third instars after seven days. 
 
Compounds Concentration (?g/g) Strain N
a
 Mortality (%) ?
2
 Effect 
Cry1Ac  Gossypol Observed Expected
b
 
Cry1Ac + 
Gossypol 
15 1500 AR1 96 92.97 37.70 81.02 Synergistic 
7.5 750  96 22.60 15.67 2.13 Additive 
3.75 375  96 6.66 8.32 0.33 Additive 
15 1500 SC1 96 98.92 85.24 2.20 Additive 
7.5 750  96 68.16 61.78 0.66 Additive 
3.75 375  96 36.56 35.17 0.05 Additive 
Cry1Ac +  
Cotton 
powder (4%) 
15 0.006 AR1 222 99.23 22.01 271.04 Synergistic 
15 0.006 SC1 221 95.60 85.15 1.28 Additive 
Cry1Ac +  
Corn powder 
(4%) 
15 NP
c
 AR1 48 47.21 14.75 71.47 Synergistic 
15  SC1 48 95.35 84.80 1.31 Additive 
a
Number of insects tested; 
b
Expected mortality is calculated from observed mortalities in 
different treatments after adjusting for control mortality. 
c
gossypol is not present in corn 
and hence not quantified. 
 
186 
 
Fig 1. Quantity of Cry1Ac protein (?g Cry1Ac/g lyophilized tissue) expressed in Bt 
cotton squares using ELISA. R1-3 = replicates 1-3. Squares were harvested July 18-Aug 
25, and squares were refrigerated at 4-7 
0
C until fed to H. zea from July 20 ? Aug. 29. 
 
 
  
 
18-Jul 29-Jul 4-Aug 18-Aug 25-Aug
Date of sampling or feeding to insects
20
-
J
u
l
24
-
J
u
l
27
-
J
u
l
30
-
J
u
l
2-
Aug
5-
Aug
7-
Aug
8-
Aug
9-
Aug
11
-
A
u
g
13
-
A
u
g
14
-
A
u
g
15
-
A
u
g
16
-
A
u
g
17
-
A
u
g
18
-
A
u
g
20
-
A
u
g
22
-
A
u
g
23
-
A
u
g
25
-
A
u
g
26
-
A
u
g
28
-
A
u
g
29
-
A
u
g
Cr
y
1
Ac
 (
?
g/
g of
 squar
e t
i
ss
ue)
0
5
10
15
20
25
30
35
R1
R2
R3
 
187 
 
 Fig 2. Cumulative % mortality of susceptible (SC) and Cry1Ac-resistant (AR) H. zea on 
Bt (DPL-555) and NBt (DPL-491) cotton squares. 
 
 
 
Days
1 4 7 101316192 252831
%
 C
u
mu
l
a
t
i
v
e
 mo
r
t
a
l
i
t
y
0
20
40
60
80
100
SC-NBt 
SC-Bt 
AR-NBt 
AR-Bt 
 
 
  
188 
 
Fig 3. Concentration of gossypol in Cry1Ac-resistant (AR1) and a susceptible (SC1) H. 
zea. A: when fed on different concentrations of gossypol (375, 750 & 1,500 ?g/g) in the 
diet alone and in 1:1combination with Cry1Ac (3.75, 7.5, 15 ?g/g). CPG = Cry1Ac + 
Gossypol; Gos=Gossypol. B: when fed on 4% cotton powder alone or in combination 
with 15 ?g Cry1Ac/g of diet.  The data represent the mean of three replications and 
standard errors are back-transformed values from logarithmic scale. 
Untreated diet Cotton powder Cry1Ac + Cotton powder
G
o
ssypo
l
 concent
r
a
t
i
on i
n
 l
a
r
v
ae 
(
ng/
m
g
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR1 
SC1 
Gossypol concentration in diet (?g/g)
0 200 400 600 800 1000 1200 1400 1600
G
o
ssyp
ol
 concent
r
a
t
i
on 
i
n
 l
a
r
v
ae 
(
ng/
m
g
)
-1
0
1
2
3
4
5
6
AR1-CPG 
AR1-Gos 
SC1-CPG 
SC1-Gos 
A
B
 
189 
 
Fig 4. Effect of 4% cotton/corn powder in the presence and absence of 15 ?g Cry1Ac/g 
of diet on larval weight in Cry1Ac-resistant (AR1) and a susceptible (SC1) H. zea. 
 
Untreated diet Corn Cry1Ac + Corn Cotton Cry1Ac + Cotton Cry1Ac
Lar
va
l
 w
e
i
g
h
t
 (
m
g)
0
20
40
60
80
100
AR1 
SC1