Genetic and Biochemical Characterization of Field-Evolved Resistance to Bacillus thuringiensis Toxin Cry1Ac in the Diamondback Moth, Plutella xylostella

Abstract

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F 1 progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with 125 I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.

Novel Isolate of Bacillus thuringiensis subsp. thuringiensis That Produces a Quasicuboidal Crystal of Cry1Ab21 Toxic to Larvae of Trichoplusia ni

“…For example, field populations of the diamondback moth, Plutella xylostella, and the cabbage looper, Trichoplusia ni, have already developed high levels of resistance due to repeated exposure to formulations based on HD1 (11,26). Furthermore, in laboratory selection studies, resistance to B. thuringiensis has evolved in the pink bollworm, Pectionphora gossypiella, and the tobacco budworm, Heliothis virescens (13,31).…”

Bacillus thuringiensis Vip3Aa Toxin Resistance in Heliothis virescens (Lepidoptera: Noctuidae)

“…Other populations resistant to Cry toxins have also been shown to exhibit polygenic resistance: for example, populations of H. virescens (46) and H. armigera (47) resistant to Cry2A and populations of P. xylostella (32) and P. gossypiella (48) resistant to Cry1Ac. Nevertheless, monogenic resistance has been found in other populations of H. virescens (37), H. armigera (41), P. xylostella (49), and Ostrinia nubilalis (50).…”

Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac

“…One involves changes in the binding of toxins to gut receptors. For example, decreases in toxin binding have been reported in resistant strains of P. xylostella (5,28). Slower activation, faster degradation (7), differential proteolytic activation (16), and sequestration by proteases (21) have all been described as potential alternative mechanisms of resistance to B. thuringiensis toxins.…”

“…Specific activities of the labeled proteins were 125 I-labeled Cry1Ab ( 125 I-Cry1Ab; 0.48 mCi/mg), 125 I-Cry1Ac (0.08 mCi/mg), and 125 I-Cry1Ca (0.02 mCi/mg) as determined by an enzyme-linked immunosorbent sandwich assay (39). Binding experiments were performed as described previously (28). Cry1Ca binding experiments were performed as a control to check qualitatively the function of the BBMVs in both populations.…”

“…Analyses with brush border membrane vesicles (BBMV) from 1 g (approximately, 250 larvae) of whole Cry1Ab-SEL and LAB-UK lastinstar larvae were conducted as described by Sayyed et al (28). Cry1Ab was 125 I labeled by a chloramine-T method (38).…”