SUMMARY OF THE ARTICLE:

- The paper discusses the genetic and biochemical basis of resistance to

Bacillus thuringiensis (Bt) toxins in diamondback moths. Three resistant
strains of diamondback moth, NO-QA from Hawaii, PEN from
Pennsylvania, and PHI from the Philippines, were compared to understand
the differences in their resistance mechanisms and inform resistance
management strategies. The study found that the NO-QA and PEN strains
showed recessive inheritance of resistance to all three Cry1A toxins, while
the PHI strain showed recessive inheritance of resistance only to Cry1Ab.
There were also family-by-toxin interactions observed, indicating the
presence of additional loci influencing resistance in the NO-QA and PEN
strains. The study also examined binding assays of the Bt toxins and found
some qualitative differences between the strains, although further
analysis is required to fully understand these differences. Overall, the
findings suggest that resistance to Bt toxins in diamondback moths can
arise independently in different populations.

MATERIALS AND METHODS:

- 1. Resistant strains of diamondback moth isolated from Hawaii (NO-QA),

Pennsylvania (PEN), and the Philippines (PHI).
- 2. Bioassays: Adult survivors of exposure to Bt toxins were used in single-pair crosses
and the F1 progeny of survivors were tested in bioassays to determine their
susceptibility to the toxins.
- 3. Binding experiments: Susceptible LAB-V strain was used in binding experiments as
an internal standard. Cry1A toxins were labeled with [125I]NaI and binding assays
were conducted using brush border membrane vesicles (BBMV) from diamondback
moth larvae.
- 4. Cry toxin preparations: Cry1A protoxins were obtained from recombinant
Escherichia coli. Liquid formulations containing Cry1C and Cry1F toxins expressed in
and encapsulated by transgenic Pseudomonas fluorescens were used. Cry1J toxin
was obtained from Ecogen strain EG7279.
- 5. Analysis of variance (ANOVA): Two-way ANOVA was performed on the mortality
data from split broods of single-pair families to test for significant variation in
mortality among families, among toxins, and family-by-toxin interactions.
- 6. Complementation tests: To determine if the loci responsible for resistance to Bt
toxins varied among populations, complementation tests for allelism were performed
by testing the offspring of single-pair crosses from each pairwise combination of
resistant strains.
- 7. Cry1Ab exposure: Larvae from the PHI strain from the Philippines were exposed to
Cry1Ab during the study to investigate the effects on resistance. These methods were
used to compare and analyze the genetic and biochemical basis of resistance to Bt
toxins among different strains of diamondback moth.
RESULTS:
The results of the study indicate that there are key differences among strains of
diamondback moth in their genetic and biochemical basis for resistance to Bt
toxins. The study compared resistant strains of diamondback moth isolated from
Hawaii, Pennsylvania, and the Philippines. The researchers conducted bioassays
on single-pair families to test for significant variation in mortality among families,
among toxins, and family-by-toxin interactions. The results showed that there
was significant genetic heterogeneity within some of the strains, indicating that
resistance to the toxins is controlled by a single locus or tightly linked loci. The
researchers also evaluated maternal effects and sex linkage by comparing the
results of reciprocal crosses, and found that resistance was autosomally
inherited. In terms of the specific toxins tested, the results revealed that the NO-
QA and PEN strains were extremely resistant to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F,
and Cry1J. They showed reduced binding of Cry1Ab and Cry1Ac, suggesting that
reduced binding of the toxins to midgut membranes conferred resistance. The
PHI strain showed resistance to Cry1Aa, but the mortality caused by the toxin
varied among the F1 families, indicating the presence of at least one dominant
mutation conferring resistance. The mortality caused by Cry1Ac in the PHI strain
ranged from 21.1% to 90%, suggesting control by one or more semidominant
mutations. Overall, the results of the study highlight the importance of
understanding the genetic and biochemical basis of resistance in diamondback
moth strains, as it can have profound implications for designing effective
resistance management strategies.

Figures in the text represent the mortality rates of different

strains of diamondback moth when exposed to various Bt
toxins. Figure 1 shows the mortality rates of different
strains when exposed to Cry1Aa, Cry1Ab, and Cry1Ac
toxins. The bars indicate the mean mortality rate and the
error bars represent the standard error. The results show
that there are differences in susceptibility to the different
toxins among the strains. For example, the PHI strain from
the Philippines shows resistance to Cry1Aa, but not to
Cry1Ab or Cry1Ac. In contrast, the NO-QA strain from
Hawaii and the PEN strain from Pennsylvania show
resistance to all three toxins. The differences in mortality
rates among the strains indicate that the genetic and
biochemical basis of resistance to Bt toxins varies among
strains. The binding assays conducted in the study also
show that the resistant strains have reduced binding of
Cry1Ab, indicating that reduced binding of toxin to midgut
membranes confers resistance to Cry1Ab in these strains.
Overall, the figures provide visual representation of the
mortality rates and genetic correlations of different strains
of diamondback moth when exposed to Bt toxins. These
findings are important for understanding the mechanisms
of resistance to Bt toxins and for developing effective
resistance management strategies.
PRESENTATION:
Title: Evolution of Resistance to Bacillus thuringiensis Toxins in Diamondback
Moth: Multiple Populations and Different Genetic Solutions
Introduction: - The paper discusses the evolutionary adaptation of
diamondback moth (Plutella xylostella) populations to Bacillus thuringiensis (Bt)
toxins. - The researchers investigate whether separate populations have arrived
at the same genetic solution when faced with similar selection pressures. - The
study aims to provide insights into the genetic and biochemical basis of
resistance to Bt toxins.
Methods: - Three resistant strains of diamondback moth were used: NO-QA
(Hawaii), PEN (Pennsylvania), and PHI (Philippines), all derived from field
populations that had been repeatedly sprayed with Bt. - Bioassays were
conducted to test the response of each strain to various Bt toxins. - Binding
experiments were performed to assess the affinity of each strain to the Cry1Aa,
Cry1Ab, Cry1Ac, Cry1C, Cry1F, and Cry1J toxins. - Genetic correlations and the
number of loci involved in resistance were determined.
Results: - The NO-QA and PEN strains showed reduced binding of Cry1Ab,
indicating that reduced toxin binding conferred resistance to Cry1Ab in these
strains. - Genetic correlations were observed between resistance to Cry1Aa,
Cry1Ab, Cry1Ac, and Cry1F in the NO-QA and PEN strains, suggesting that a single
locus mutation confers resistance to these four toxins. - In contrast, resistance to
Cry1F in the PHI strain was not correlated with resistance to the Cry1A toxins. -
Lack of specific binding was associated with resistance to Cry1Ac in NO-QA and
PEN but not in PHI, implying different genetic mechanisms among the strains.

 (Populations from Hawaii and Pennsylvania have a genetic locus associated with
reduced toxin binding and high resistance to four Bt toxins.

 Resistance in a population from the Philippines is controlled by multiple loci and has a
narrower spectrum.

 In the Philippines population, resistance to some Bt toxins is not recessive and not
associated with reduced binding.

Discussion: - The findings suggest that resistance to Bt toxins in diamondback

moth populations has evolved independently in Hawaii and Pennsylvania. - The
results highlight the importance of considering variation in resistance traits
among populations when designing resistance management strategies. - The
study provides valuable insights into the genetic and biochemical basis of
resistance to Bt toxins in diamondback moth.
Conclusion: - The study demonstrates multiple genetic solutions to resistance in
diamondback moth populations exposed to Bt toxins. - The findings emphasize
the need for comprehensive resistance management strategies that take into
account population-specific variation in resistance traits. - Further research is
needed to explore the genetic mechanisms underlying resistance to different Bt
toxins and to develop effective resistance management strategies.

The researchers conducted experiments to compare resistant strains of the

diamondback moth from Hawaii, Pennsylvania, and the Philippines to determine
the genetic and biochemical basis of resistance to Bacillus thuringiensis (Bt)
toxins.
The goal of the study was to provide insights for resistance management
strategies in pest control.
The researchers used bioassays to test the resistance of the diamondback
moth larvae to various Bt toxins. THEY FOUND THAT;
- Pennsylvania (PEN) strain showed strong genetic correlations in resistance
to four different Cry1A toxins, suggesting that a single mutation at a
specific locus conferred resistance.
- Philippines (PHI) strain showed resistance to Cry1Ab but not to the other
three Cry1A toxins, indicating that mutations at multiple loci were involved
in resistance.
- To further investigate the genetic basis of resistance, the researchers
performed complementation tests and found that the same locus
controlled Cry1Ab resistance in both the Hawaii (NO-QA) and Pennsylvania
strains, but not in the Philippines strain.
- The researchers concluded that multiple mutations were necessary in the
Philippines strain to confer resistance to the three Cry1A toxins and an
additional mutation was required for resistance to Cry1F.
- In terms of the biochemical basis of resistance, the researchers conducted
binding studies to analyze the interaction between the Bt toxins and the
brush border membrane vesicles of the diamondback moth. They found
that the resistant strains showed lack of specific binding to Cry1Ac and
Cry1Ab, but not to Cry1Aa. However, the researchers cautioned that these
binding results should be interpreted cautiously due to the limited
resolution of the technique used. Overall, the study demonstrates that
there is genetic and biochemical variation in the resistance of
diamondback moths to Bt toxins among different strains. This finding
highlights the importance of considering this variation in designing
resistance management strategies.
 -
Diamondback moth is the first insect to evolve resistance to bt in open-field
populations.
- Populations from hawaii and pennysylvania share a genetic locus at which a recessive
mutation associated with reduced toxin binding confers extremely high resistance to
four bt toxins which are 3 cry1a and cry1f.
- In contrast, resistance in a population from philippines shows multilocus control , a
narrower spectrum and for some bt toxins inheritance is not recessive and not
associated with reduced binding.
- ACTING MECHANISMS OF THOSE TOXINS: It kills insects by binding and creating pores
in midgut membranes.
- The study compared resistant strains of diamondback moth isolated from
Hawaii (NO-QA), Pennsylvania (PEN), and the Philippines (PHI).
- The researchers used family mean correlations to estimate genetic correlations
of resistance between pairs of toxins.
- Strong genetic correlations were observed in resistance between all six pairwise
combinations of Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F in the PEN strain.
- Significant family-by-toxin interactions were observed in mortality of split
broods from the PEN and NO-QA strains, suggesting the presence of additional
loci influencing resistance in these strains.
- Mortality caused by Cry1Ac to the F1 families from the PHI strain ranged from
21.1 to 90%, indicating control by one or more semidominant mutations.
- Complementation tests showed that resistance to Cry1Ab in the PHI strain is
controlled at least in part by allelic variation at the same locus that controls
multitoxin resistance in NO-QA and PEN.
- The presence of a multitoxin resistance gene in both the NO-QA and PEN
strains was indicated by reduced binding to a shared binding site.
- The presence of one or more additional loci influencing resistance in the NO-QA
and PEN strains was suggested by significant family-by-toxin interactions in
mortality.
- The presence of one or more semidominant mutations influencing resistance in
the PHI strain was indicated by the range of mortality caused by Cry1Ac.

The common soil bacteriumBacillus thuringiensis(Bt) produces crystals containing insecticidal proteins (1). These toxins kill insects by binding to and creating pores in midgut membranes (2). Each of the many strains of Bt
produces a characteristic set of cr ystal proteins (1). Each of these proteins is extremely toxic to certain insects yet is harmless to most other organisms, including people, wildlife, and even other insects
(1). Because of their highly specific toxicity, Bt toxins offer tremendous benefits for insect pest management. Populations of more than 500 species of insects and mites
have evolved resistance to insecticides and acaricides (3);laboratory selection experiments show that many pests can also adapt to Bt toxins (4). So far, documented cases of resistance
to Bt in open-field populations of pests are limited to one insect species, the diamondback moth (Plutella xylostella) (4). However, greatly increased use of Bt toxins, whether delivered by conventional sprays or by
genetically engineered crops, raises the likelihood that pests will adapt (4 – 6). Transgenic corn and cotton that express the lepidopteran-active Bt toxins Cr y1Ab and Cr y1Ac, respectively, were grown commercially in the
United States for the first time during 1996. In 1997, Bt expressing corn, cotton, and potatoes were planted on more than 3 million hectares in the United States (7). This major increase in exposure to Bt toxins intensifies
selection for resistance in pests.
The goal of resistance management is to delay resistance in pests; success requires knowledge about the genetic and biochemical basis of resistance (8). For example, the widely recommended refuge/high-dose strategy is
based on the idea that refuges from exposure to Bt permit susceptible insects to survive and mate with resistant insects emerging from nearby Bt crops. This strategy is expected to work best if resistance is
recessive, so that hybrid offspring from resistant and susceptible parents are killed by the toxin in the Bt-expressing crop. Previously reported evidence shows that in some strains of diamondback moth, resistance to some Bt
formulations and toxins is inherited as a partially or completely recessive trait (11–15). The resistant NO-QA strain of diamondback moth from Hawaii harbors a recessive mutation that confers resistance to at least four Bt
toxins (14). Although various mechanisms of resistance are possible (13, 16, 17), reduced binding of toxin to midgut membranes is the only mechanism of resistance to Bt known in diamondback moth.A key question in
resistance management and evolutionary theory is whether separate populations arrive at the same genetic solution when faced with similar selection (18, 19). If this occurs, either by gene f low that spreads adaptive muta-
tions (20) or by independent evolution, optimal strategies for managing resistance can be devised by analyzing a few resistant populations. We tested this hypothesis by comparing resistant strains of diamondback moth
isolated from Hawaii (NO-QA), Pennsylvania (PEN), and the Philippines (PHI). Previous comparisons among independently published studies suggested that differences among strains might occur (15, 21, 22), yet it was not
clear if the obser ved variation was genetically based or caused by variation in environmental conditions and techniques among laboratories. Direct experimental comparisons reported here revealed key differences among
strains of diamondback moth in the genetic and biochemical basis of resistance to Bt toxins that profoundly affect the choice of resistance management strategies.

The study i would like to talk about today is about the variations in the genetic and
biochemical basis of diamondback moth resistance to bacillus thuringiensis. Bacillus
thuringiensis is a soil bacterium that produces toxins that are killing certain insects and
meanwhile safe for other organisms including humans. These toxins are able to kill the
insects by binding and creating pores on midgut membrane. The challenge is the potential
for pests to develop rapid resistance to bt toxins and in order to manage this, researchers
decided to undersand the resistance mechanism and if it varies between global populations.

Researchers developed a strategy based on the idea of refuges which provides a safe space
for susceptible insects to survive and mate with resistant ones emerging from nearby bt
crops. This approach works best if the resistance is recessive, meaning that hybrid offspring
from resistant and susceptible parents are still susceptible to the bt toxin in the crop.
However, research has shown that the genetic and biochemical basis of resistance in
different strains of pests can vary, which complicates the development of effective
resistance management strategies.

Diamondback moth is the first insect that shows resistance to bt toxins in open-field
populations.