Source: N Y AGRICULTURAL EXPT STATION submitted to NRP
FUNCTIONAL CHARACTERIZATION OF KNOCKDOWN RESISTANCE MUTATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0189378
Grant No.
2001-35302-10880
Cumulative Award Amt.
(N/A)
Proposal No.
2001-02784
Multistate No.
(N/A)
Project Start Date
Oct 1, 2001
Project End Date
Sep 30, 2003
Grant Year
2001
Program Code
[(N/A)]- (N/A)
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
ENTOMOLOGY
Non Technical Summary
Knockdown resistance to pyrethroid insecticides, which involves reduced sensitivity of the insect nervous system to these compounds, threatens the continued use of pyrethroids to control agricultural pests and vectors of human and animal diseases. Knockdown resistance is genetically linked to insect voltage-sensitive sodium channel genes, and at least 18 different sodium channel gene mutations have been associated with resistance in various species. The long-term objective of this research program is to define the molecular basis of knockdown resistance. The proposed studies, based conceptually and technically on our prior research that defined the molecular basis of knockdown resistance in the house fly model system, will define the functional significance of sodium channel gene mutations that are associated with knockdown resistance traits in other pest species and to assess the impact of these mutations on the sensitivity of insect sodium channels to other insecticidal agents.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110100025%
2113110104025%
2113110113025%
2113110118025%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology; 1180 - Pharmacology; 1000 - Biochemistry and biophysics;
Goals / Objectives
This research project addresses the problem posed by the development of resistance to pyrethroid insecticides arising from a mechanism known as kdr (knockdown resistance), which is caused by nerve insensitivity. This type of resistance has been genetically linked to molecular markers lying within insect voltage-sensitive sodium channel genes, the molecular target sites of pyrethroids, and numerous resistance-associated mutations have been described. The objectives of the proposed research are to determine whether these resistance-associated mutations alters the sensitivity of insect sodium channels to pyrethroids and they also modify the sensitivity of sodium channels to other insecticidal agents acting at this site.
Project Methods
The principal approach involves the functional expression of native and specifically mutated house fly sodium channels in cells and the electrophysiological analysis of the pharmacological properties of the expressed channels. Wildtype and specifically mutated house fly sodium channels will be expressed in oocytes of the frog Xenopus laevis, and the properties of the expressed channels will be determined by measuring sodium currents by two-electrode voltage clamp.

Progress 10/01/01 to 09/30/03

Outputs
This project examines the impact of sodium channel gene mutations identified in pyrethroid-resistant strains of various insect species on the sensitivity of pyrethroid, pyrazoline, and other insecticides on house fly sodium channels expressed in Xenopus oocytes. Progress toward the completion of project objectives was achieved in the following areas. Additional specifically mutated house fly sodium channels were constructed for use in functional expression assays. Efforts were also initiated to construct clones representing splice variants of this sequence that are thought to exhibit differential sensitivity to pyrethroid insecticides. Electrophysiological methods were developed to investigate the action of pyrazoline insecticides on sodium channels expressed in oocytes. These studies documented the state-dependent modification of both house fly and rat skeletal muscle sodium channels by the bioactivated metabolite of indoxacarb and the experimental pyrazoline RH 3421. Additional progress was made in the expression of insect sodium channels in insect cell lines. We employed a specially-constructed vector to transform Drosophila S2 cells with the house fly sodium channel alpha subunit cDNA under the control of the Drosophila HSP70 promoter, and we used a commercial vector to transform cells with the house fly auxiliary subunit under the control of the Drosophila metallothionein promoter. Of these, only the cell line expressing the auxiliary subunit demonstrated promoter-driven over-expression of the transgene. Modification of the commercial vector to accept the alpha subunit cDNA and the construction of this clone were in progress at the end of the project period.

Impacts
The molecular characterization of knockdown resistance in insects contributes to the development of rational resistance management strategies to delay or overcome resistance to pyrethroids and other sodium channel-directed insecticides in pest populations.

Publications

  • Lee, S. H., Ingles,P. J., Knipple, D. C., and Soderlund, D. M. 2002. Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene. Invert. Neurosci. 4: 125-133.
  • Soderlund, D. M. and Knipple, D.C. 2003. The molecular biology of knockdown resistance. Insect Biochem. Mol. Biol. 33: 563-577.
  • Soderlund, D. M. 2003. Sodium channels. In "Comprehensive Insect Physiology, Biochemistry, Pharmacology and Molecular Biology" (L. I. Gilbert, K. Iatrou and S. Gill, Eds.), Elsevier, New York (in press).


Progress 01/01/02 to 12/31/02

Outputs
This project examines the impact of sodium channel gene mutations identified in pyrethroid-resistant strains of various insect species on the sensitivity of pyrethroid, pyrazoline, and other insecticides on house fly sodium channels expressed in Xenopus oocytes. Efforts during this year focussed on three aspects of the research: (1) the construction of new specifically mutated house fly sodium channel cDNAs; (2) studies of the action of pyrazoline insecticides and related compounds on insect sodium channels expressed in oocytes; and (3) continued efforts to develop transformed insect cell lines that express wildtype or specifically mutated insect sodium channels.

Impacts
The molecular characterization of knockdown resistance in insects contributes to the development of rational resistance management strategies to delay or overcome resistance to pyrethroids and other sodium channel-directed insecticides in pest populations.

Publications

  • Lee, S. H., Ingles,P. J., Knipple, D. C., and Soderlund, D. M. 2002. Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene. Invert. Neurosci. 4: 125-133.


Progress 01/01/01 to 12/31/01

Outputs
This new project, initiated 10/1/01, examines the impact of sodium channel gene mutations identified in pyrethroid-resistant strains of various insect species on the sensitivity of pyrethroid, pyrazoline, and other insecticides on house fly sodium channels expressed in Xenopus oocytes. Progress to date, since the inception of the project has been limited to the construction of new specifically mutated house fly sodium channel cDNAs and the development of electrophysiological methods to investigate the action of pyrazoline insecticides on sodium channels expressed in oocytes.

Impacts
The molecular characterization of knockdown resistance in insects contributes to the development of rational resistance management strategies to delay or overcome resistance to pyrethroids and other sodium channel-directed insecticides in pest populations.

Publications

  • No publications reported this period