Discipline: Arthropod management

Konasale J. Anilkumar¹, Ana Rodrigo-Simón², Juan Ferré², Marianne Pusztai-Carey³ and William J. Moar¹

Author affiliation:

¹Department of Entomology, Auburn University, Auburn, AL 36849

²Department of Genetics, University of Valencia, Dr. Moliner 50, 46100 Burjassot (Valencia),

Spain

³Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue,

Cleveland, OH 44106-4935

Corresponding author:

William J. Moar

301 Funchess Hall

Department of Entomology and Plant Pathology,

Auburn University, Auburn, AL-36849

Ph: 13348442560

Fax: 13348445005

Email: moarwil@auburn.edu

Acknowledgements

This work was partially supported by United States Department of Agriculture- Southern Insect Management Research Unit (USDA SIMRU), Stoneville, MS, Cotton Incorporated, Cary, N.C and the Spanish Ministry of Education and Science (projects No. AGL2003-09282-C03-01 and AGL2006-11914).

Production and characterization of Bt Cry1Ac resistance in cotton bollworm, Helicoverpa zea (Boddie)

Abstract

Cry1Ac-resistant Helicoverpa zea populations were established by selection with Cry1Ac toxin (AR) or MVP II (MR). The resistance ratio (RR) for AR reached >100-fold whereas MR crashed after 11 generations. AR had significant but low levels of cross-resistance to MVP II. AR was cross-resistant to Cry1Ab, but not cross-resistant to other Bt proteins and cypermethrin. Binding assays showed no significant differences. These results help explain why this species has not evolved Bt resistance.

Key Words: Bacillus thuringiensis, Cry1Ac resistance, Cross-resistance, Helicoverpa zea

Introduction

Cotton bollworm (CBW), Helicoverpa zea is one of the major insect pests of cotton in the U. S. and is one of the target pests of Bollgard^® that expresses the Cry1Ac insecticidal protein from Bacillus thuringiensis (Perlak et al., 1990). Though Bollgard^® provides unprecedented control against tobacco budworm (TBW) and pink bollworm (PBW), it is not economically effective against CBW post bloom and/or when the crop is under stress (Jackson et al., 2004). Additionally, Bollgard^® does not express a ‘high-dose’ in all plant parts necessary to kill all CBW larvae, thereby increasing the likelihood of resistance development (Greenplate 1999, and Adamczyk et al., 2001).

Although there is no documented field resistance to Bollgard^® by any target pest, laboratory selection studies with TBW, PBW and Helicoverpa armigera have indicated the inherent capacity of insects to adapt to Cry1Ac. These results have contributed to insect resistance management (IRM) policy making, but this has not happened with CBW because the study of Cry1Ac resistance in CBW have not been as successful as for other target pests. Because each species can have different resistance characteristics (Akhurst et al., 2003, Morin et al., 2003, Bird and Akhurst, 2004, Tabashnik et al., 2003, 2004, and Xie et al., 2005), potentially impacting resistant management strategies (Bates et al., 2005), characterizing Cry1Ac resistance in CBW is critical. Additionally, selection experiments cited above were conducted with MVP II, a commercial formulation containing Cry1Ac protoxin inclusion bodies (Gould et al., 1995). Furthermore, because Bollgard^® expresses solubilized Cry1Ac protoxin that is at least partially activated to toxin upon ingestion; we hypothesize that selection using MVP II may not adequately reflect resistance selection occurring in planta. Therefore, we initiated selection experiments using MVP II and Cry1Ac toxin to compare resistance characteristics using these two different selection pressures.

Materials and Methods

Selection experiments: Two strains of CBW were selected for Cry1Ac resistance through diet incorporation of Bt proteins. Individual neonates were exposed to MVP II (MR) or Cry1Ac toxin (AR) for 7d; only those larvae that molted were selected and reared to pupation on diet containing no Bt protein.

Testing resistance and cross-resistance: At selected generations, diet incorporation bioassays were conducted concurrently for lab culture (LC) and resistant strains to determine levels of resistance. Individual neonates were tested and assays were rated after 7D. Bioassays were replicated at least three times and data were analyzed by probit analysis using POLO-plus. Dead and first instar larvae were considered as dead and included in the calculation of lethal concentrations. LC₅₀ values with non-overlapping of 95% confidence limits were considered as significantly different. Tests for cross-resistance to other Cry proteins (Cry1Ab, MVP II, and Cry2A), and cypermethrin were conducted between generations 15 and 20 of selection (RR>100-fold). Topical bioassays were conducted with third instar larvae following a modified (Usmani and Knowles, 2001) method for testing cross-resistance to cypermethrin. Twelve larvae were tested per concentration; treated larvae were transferred to 24 well bioassay trays containing diet. Mortality was assessed after 24 h and assays were replicated four times. Lethal doses were calculated using probit analysis and adjusted for body weight.

Labeling of Cry1Ac toxin: Bt Cry1Ac toxin was produced and labeled as described by Estela et al. (2004) and Van Rie et al. (1989).

BBMV preparation and binding assays: Fifth instar larvae were dissected in MET buffer and midguts were removed and frozen at -80 ºC. Brush border membrane vesicles (BBMV) were prepared by the differential magnesium precipitation method, frozen in liquid nitrogen and stored at -80 ºC until used.

A fixed amount of ¹²⁵I-labeled-toxins and BBMV (0.05 mg/ml) were incubated for 1 h at room temperature with increasing concentrations of unlabeled homologous toxin in 0.1 ml final volume of binding buffer. After 10 min 16000 x g centrifugation, pellets were washed twice in binding buffer. The final radioactivity remaining in the BBMV pellets was measured. Experiments were replicated three times.

Results and Discussion

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 strain (LC). The rate of resistance evolution in AR increased with an increase in selection pressure and 12, 36 and 123-fold resistance was observed after 4, 7 and 11 generations of selection, respectively. The laboratory strain originating from Monsanto has had annual infusions of CBW collected from corn and therefore should have higher genetic variability than laboratory colonies with no infusion of field derived insects. Resistance in MR did not increase above 17-fold. Selection in MR could not be continued beyond 8 generations ultimately leading to loss of the strain after 11 generations. The loss of MR after achieving only low level resistance is contrary to reports for other insects such as PBW and TBW (Gould et al. 1995, Tabashnik et al. 2003). However, our current results with CBW agree with previous unpublished observations by at least two different laboratories. Furthermore, concurrent selection with the same parental colony resulting in moderately high and stable resistance to Cry1Ac toxin but not to MVP II further validates prior reports.

Cross-resistance of AR to Bt proteins and cypermethrin. MVP II was more toxic to AR than expected, resulting in low but significant cross resistance. Only partial cross resistance in AR to MVP II suggests further that MVP II may not be the appropriate Cry1Ac selection agent against H. zea. There was significant cross-resistance to Cry1Ab. This is not unexpected because Cry1Ab and Cry1Ac toxins share greater than 90% amino acid homology (Crickmore et al. 1998). However, this cross-resistance is unlikely to be related to changes in binding affinity of Cry1A toxins because no binding differences were observed between LC and AR (discussed below). There was no cross resistance to Cry2A. This was probably due to differences in amino acid sequence and mode of action between Cry1Ac and Cry2A (English et al. 1994). Cry1Ac-resistant TBW (YHD2), PBW and H. armigera also have shown no detectable cross resistance to Cry2A proteins (Akhurst et al. 2003, Gould et al. 1995, Tabashnik et al. 2003). Cypermethrin was tested for cross resistance in AR because growers often spray Bt cotton with pyrethroids when high CBW populations exist, and pyrethroid oversprays are currently recommended to mitigate CBW resistance to Bt cotton. No cross-resistance was observed for AR topically applied with cypermethrin. These results would suggest that pyrethroids can continue to be used when necessary. Additionally, because pyrethroids have been used against this pest annually since the adoption of Bt cotton in 1996, pyrethroid use might have helped to reduce resistance evolution to Bt cotton by CBW.

Binding of ¹²⁵I-labeled Cry1A toxins to BBMV: The narrow spectrum of Bt resistance suggests an alteration in the Cry1Ac binding site, and lack of Cry1Ac binding has been reported in some Cry1Ac resistant populations of TBW, PBW, and H. armigera (Akhurst et al. 2003, Gould et al. 1995, and Tabashnik et al. 2003). However, binding of ¹²⁵I-Cry1Ac to BBMV from AR and LC did not show significant differences suggesting that a reduction in binding is not the mechanism of Cry1Ac resistance in AR. Therefore, 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).

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