Source: KENTUCKY STATE UNIVERSITY submitted to

RESISTANCE OF STORED GRAIN MOTH PESTS TO TRANSGENIC GRAIN

PROJECT DIRECTOR: SEDLACEK, J. D. PERFORMING ORGANIZATION
PLANT & SOIL SCIENCES
KENTUCKY STATE UNIVERSITY
FRANKFORT,KY 40601 NON TECHNICAL SUMMARY: Transgenic Bt grain is
being stored on-farm in corn producing states without knowing the impact it has on
stored grain moth pest populations or resistance potential of these pests. This project
examines resistance potential and mechanisms of resistance of the moths and will
propose resistance management strategies. OBJECTIVES: The overall purpose of this
study is to investigate resistance potential and mechanisms of resistance for Indian meal
moth (IMM) and Angoumois grain moth (AGM) to transgenic (Bt transformed) grain.
Specifically, we will 1) quantify resistance potential of Kentucky and Kansas susceptible
and resistant populations of IMM and AGM to transgenic grain, 2) determine
physiological mechanisms of resistance in IMM and AGM to transgenic grain, and 3)
assess potential of field populations of IMM and AGM in on-farm stored corn to develop
resistance. APPROACH: Specifically, we will examine Kentucky and Kansas
populations of IMM and Kentucky populations of AGM for resistance development and
mechanisms of resistance to transgenic grain that has a single or multiple gene(s)
encoding Bacillus thuringiensis (Bt) insecticidal toxin production. Populations of moth
species from different geographic locations exhibit different susceptibilities/resistance
factors to Bt (Tabashnik 1994). Examining resistance of these insects to Bt grain will
ultimately lead to developing resistance management strategies and more economical and
environmentally sound management of stored grains not only in Kentucky, but nationally
and internationally. All laboratory assays will be performed in the Stored Grain Insect
Management and Ecology Laboratory at Kentucky State University while field
investigations will be conducted at the stored grain research complex on the Kentucky
State University Agricultural Research Farm. Training for determining mechanisms of
resistance will take place at the GMPRC in Manhattan, KS. Susceptible Kentucky IMM
and AGM colonies are reared in my laboratory on ground wheat diet or maize,
respectively. Each colony was started with individuals collected from grain bins on the
Kentucky State University Agricultural Research Farm in 1995. The GMPRC will
provide Bt susceptible and resistant IMM colonies from Kansas. Monsanto will provide
purified Cry1Ab Bt toxin, and has already provided grain of DeKalb 679 BtY (an isoline
of transgenic corn expressing the Cry1Ab toxin), grain of DeKalb 679 (its Bt- isoline);
both of which are commonly grown in Kentucky. Monsanto will also provide purified
Monsanto Proprietary Gene 2 (MPG 2) toxin, grain of the yet to be released hybrid
expressing MPG 2 toxin, Mon 810 + MPG 2 expressing Cry 1Ab and MPG 2 toxins, and
their non Bt isoline (LEPOTD19). Dow AgroSciences will provide purified Cry1F toxin,
grain of the yet to be released corn hybrid expressing Cry 1F toxin and its non-Bt isoline.
Purified Cry1Ab, MPG 2, Cry 1Ab + MPG 2, and Cry 1F toxins that are expressed in the
transformed corn plants will be used to establish baseline and post selection LD50 values
in order to determine the change in susceptibility for each IMM and AGM strain used.
The change in moth susceptibility will be quantified before and after 2, 5, and 10
consecutive generations of rearing on the transgenic grain. An increase in LD50 values
from pre-transgenic grain exposure will be indicative of resistance development.
Individuals from each of the IMM and AGM strains selected will be examined for
differences in toxin binding and gut proteolytic activity after recorded changes in
susceptibility. DeKalb 679 BtY will be used to assess development of resistance to
transgenic grain by field populations of IMM and AGM. Resistance will also be
examined by establishing baseline and first and second storage season LD50 values for
populations of each moth species in bins and cribs using purified Cry1Ab toxin.

CRIS NUMBER: 0190198 SUBFILE: CRIS

PROJECT NUMBER: KYX-01-11390 SPONSOR AGENCY: NIFA
PROJECT TYPE: OTHER GRANTS PROJECT STATUS: TERMINATED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Sep 15, 2001 TERMINATION DATE: Sep 30, 2005

GRANT PROGRAM: CAPACITY BUILDING GRANTS - 1890 / RESEARCH

GRANT PROGRAM AREA: Federal Administration
 CLASSIFICATION
Knowledge Area (KA) Subject (S) Science (F) Objective (G) Percent

211 1510 1130 4.2 100%

CLASSIFICATION HEADINGS
KA211 - Insects, Mites, and Other Arthropods Affecting Plants
S1510 - Corn
F1130 - Entomology and acarology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks

RESEARCH EFFORT CATEGORIES

BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%

KEYWORDS: plodia interpunctella; sitotroga cerealella; corn; transgenic plants;

bacillus thuringiensis; resistance management; stored product insects; defense
mechanisms; quantitative analysis; regional research; insect physiology; risk
assessment; bioassays; laboratory tests; cooperative research; proteolysis;
binding; baseline studies.

PROGRESS: Sep 15, 2001 TO Sep 30, 2005

This research showed that Cry 1Ab corn negatively impacted life history
parameters of IMM and AGM populations. Also it was found that Cry 1Ab+Cry
2Ab corn negatively impacted life history parameters of IMM and AGM
populations. However, the Cry 2Ab transgenic corn did not have any significant
effects on any of the IMM life history parameters, but did negatively impact AGM
duration of development. Cry 1F transgenic corn negatively impacted both the
KY and KS Dipel- susceptible and -resistant IMM populations but did not have a
significant effect on the AGM. Because Dipel- resistant populations were
negatively effected, it was demonstrated that the Cry 1F protein is not cross-
resistant with the Cry 1A proteins found in Dipel. This research also showed that
all of the IMM strains developed resistance to Cry 1F transgenic corn in four
generations. Only the KY and KS Dipel-susceptible populations developed
resistance to Cry 1Ab transgenic corn in four generations. These results suggest
that resistance to transgenic Bt crops will no necessarily be maintained because
none of the Dipel-resistant IMM populations had increased LC50 values to Cry
1Ab or Cry 2Ab lyophilized tissue which consisted of cry proteins found in Dipel.
Conclusions about resistance development to the stacked technology (Cry 1Ab+
Cry 2Ab) could not be made from this research. The mechanism by which IMM
becomes resistant to transgenic Bt corn was investigated. Binding sites and
proteolytic enzyme mechanisms of IMM resistance to topically applied Bt
products and Bt transgenic corn were studied. Reduced enzyme activity which is
thought to be due to less Bt protoxin being processed to the active toxin form by
midgut proteinases was observed in this study. Also seen was an increase in
enzyme activity in the KY Dipel-resistant populations reared on Cry 1F transgenic
corn, which was thought to be due to the lower amount of Bt protein in transgenic
corn kernels. The second mechanism of resistance, altered binding of activated
toxin to BBMV in insect midguts, was not observed. No detectable differences in
the binding of either the Cry 1Ab toxin to IMM BBMV was seen. However a major
concern was in the preparation of the BBMV. A large amount of insects was
needed for sufficient protein concentration which was difficult to manipulate once
the rearing process was initiated on the transgenic corn for four consecutive
generations. Another concern was protein degradation of the samples which I
attempted to avoid by adding proteinase inhibitor tablets and working as quickly
as possible, but protein degradation did take place. Better methods need to be
developed to collect adequate BBMV samples from small insects such as IMM so
that binding studies can be as accurate as possible. It was concluded from this
research that changes in proteolytic activity and not altered binding is the
mechanism of resistance for IMM to Bt transgenic grain. IMM taken from grain
bins containing Cry 1Ab shelled corn or its non Bt isoline were used to start
laboratory colonies. However, new laboratory personnel were unable to
successfully conduct the established LD50 bioassays at 8 doses.

IMPACT: 2001-09-15 TO 2005-09-30 Scientists recognize that evolution of

resistance is a significant threat to continued development and success of Bt
insecticides and transformed plants. Thus, information provided by these
investigations is essential to the development of tactics that may delay or reduce
the evolution of resistance to Bt corn, or for the development of novel products
that can replace those which pose resistance concerns. Proactive description of
resistance development and mechanisms of resistance will prolong the useful life
of potentially important tools for managing insect pests of stored grain and other
commodities. Further studies with different insects in laboratory and especially
field settings need to be performed in order to gain full understanding of the
mechanisms of resistance development.

PUBLICATION INFORMATION: 2001-09-15 TO 2005-09-30

Wilkins, T.M. 2004. Resistance potential and mechanisms of resistance of Plodia
interpunctella (Hubner) (Lepidoptera: Pyralidae) to several Bacillus thuringiensis
Berliner transformed corn hybrids. MS thesis, University of Kentucky, Lexington.
130 pp.

PROJECT CONTACT INFORMATION

NAME: Sedlacek, J. D.
PHONE:502-597-6582
FAX: 502-597-6381