Source: UNIVERSIDAD AUTONOMA DE NUEVO LEON submitted to
BACILLUS THURINGIENSIS RESISTANCE IN LEPIDOPTERA
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0404672
Grant No.
(N/A)
Project No.
0210-22310-002-21S
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 18, 2001
Project End Date
Apr 30, 2006
Grant Year
(N/A)
Project Director
GREENE R V
Recipient Organization
UNIVERSIDAD AUTONOMA DE NUEVO LEON
APARTADO POSTAL 414 Y 2790
NUEVO LEON,null null
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2152410110240%
2154010116030%
5111510110230%
Goals / Objectives
To study at a molecular level mechanisms of Bacillus thuringiensis (Bt) in Lepidoptera.
Project Methods
Bt resistant insects (Spodoptera frugiperda and Heliothis virescens, and other Lepidoptera as needed) will be selected by exposure of sequential generations of larvae to purified toxin. Mid-gut from insects will be used to isolate total RNA and RT-PCR will be utilized to survey for potential molecular markers of resistance. Comparative studies will be conducted using non-resistant and resistant insects with focus on the production of anti-toxin molecules.

Progress 10/01/05 to 09/30/06

Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Universidad Autonoma de Nuevo Leon (UANL). The study seeks to evaluate and characterize Bacillus thuringiensis (Bt) resistance mechanisms of selected Lepidoptera in the field and laboratory. The present report includes results of the evaluation of Trichoplusia ni strains. There were no differences found in susceptibility to Bt among strains of Heliothis virescens and Spodoptera frugiperda from field or laboratory. Research results, based on T. ni experimental data, included 1) differences in the physiology and susceptibility to Bt toxins and protoxins of tested strains, 2) the enzymatic activity patterns after exposing strains to several substrates, and 3) the antimicrobial peptides produced among strains. The study based on the differences in the physiology and susceptibility to Bt toxins and protoxins of tested strains was recently published (Tamez-Guerra P., Damas G., Iracheta M.M., Gomez-Flores R.A., Oppert B., and Rodriguez-Padilla C. 2006. Differences in susceptibility and physiological fitness of Trichoplusia ni (Hubner) strains to Bacillus thuringiensis exposure. J Econ Entomol. 99:3. 937-945). The student who was supported with this project, Gabriela Damas-Buenrostro, presented her master of sciences degree thesis defense during December 2005. The draft of the study based on the antimicrobial peptides produced among strains (Tamez-Guerra P., Alcocer-Gonzalez, Valadez, J.M., A., Oppert, B., and Rodriguez-Padilla, C. Comparison of immune response peptides of Mexican trains of Trichoplusia ni (Hubner) exposed to Bacillus thuringiensis) is currently under revision by our research team. The paper will be submitted to the Journal of Invertebrate Pathology. The student who was supported with this project, Alberto Valadez, presented his master of sciences degree thesis defense October, 2005. Differences in enzymatic activity of Mexican and US Trichoplusia ni (Hubner) strains exposed to Bacillus thuringiensis is being revised by our research team. It will be sent to Dr. Oppert for further revision. It will be submitted to the Journal of Insect Biochemical and Molecular Microbiology. Dr. Magdalena Iracheta was supported with this project as a postdoctoral research associate.

Impacts
(N/A)

Publications


    Progress 10/01/04 to 09/30/05

    Outputs
    4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Universidad Autonoma de Nuevo Leon (UANL). The study seeks to evaluate and characterize Bacillus thuringiensis (Bt) resistance mechanisms of selected Lepidoptera in the field and laboratory. Strains of Trichoplusia ni (Hubner) were collected from different locations in the bajio guanajuatense area in Mexico. These strains were maintained in the laboratory and were used in three different research projects: 1) A comparison in the physiology and susceptibility to Bt toxins and protoxins was made among different T. ni strains. Differences were observed in susceptibility to Bt formulations among T. ni populations collected in several bajio areas. Using a droplet feeding bioassay, T. ni neonates collected in Romita, Guanajuato (Gto), were more susceptible to Bt than those collected in Salvatierra or San Luis de la Paz, Gto. The impact of this study was the establishment of a baseline of susceptibility to Bt in Mexico. Laboratory selections for resistance to Bt formulations were unsuccessful and suggest that resistance alleles to Bt in these T. ni populations were low; 2) A study was completed on the digestive enzymatic activity patterns of different T. ni strains. The midgut protease activity ratios of trypsin:chymotrypsin in laboratory strains of T. ni were different for the Mexican and American laboratory strains and a Mexican field strain of T. ni isolated form Salvatierra, Gto.. However, chymotrypsin activity was always dominant, indicating that this enzyme is important in T. ni digestion. However, the relevance to Bt susceptibilities in these strains is unknown; and 3) A comparison of antimicrobial peptides in different T. ni strains was made using RT-PCR . Differences in antimicrobial peptides levels were observed among T. ni strains, based on the Bt dose, instar, and time of exposure. Most notably, even though attacin was detected only in 11% of the samples, it was detected among all instar larvae and pupae of a field strain exposed to 500 IU/mL of Xen TariRG after 20 h of Bt exposure. However, more research is needed to understand the role that antimicrobial peptides may play in Bt resistance.

    Impacts
    (N/A)

    Publications


      Progress 10/01/02 to 09/30/03

      Outputs
      4. What were the most significant accomplishments this past year? This report serves to document research conducted under a specific cooperative agreement between ARS and the Universidad Autonoma de Nuevo Leon (UANL). The study seeks to evaluate and characterize Bacillus thuringiensis (Bt) resistance mechanisms of selected Lepidoptera in the field and laboratory. Work in this reporting period focused on Trichoplusia ni (Hubner). Significant differences in various T. ni strains from the field and the laboratory to individual Bt proteins (Cry1Aa, Ab, and Ac proteins and toxins) were observed. After five generations of exposure to a commercial Bt formulation, resistance levels of T. ni increased significantly. Mid-gut protease activity (trysin:chymotrypsin) changed, but chymotrypsin was always dominant. Zymograms indicated that an additional protease was also present. Gene expression of the antimicrobial peptides cecropine A and an attacin-like protein was detected in Bt-exposed and non-exposed larvae. Tests utilizing specific primers to further characterize the expression of these peptides are underway.

      Impacts
      (N/A)

      Publications