Deletion of the Cry11A or the Cyt1A toxin from Bacillus thuringiensis subsp. israelensis: effect on toxicity against resistant Culex quinquefasciatus (Diptera: Culicidae).

Bacillus thuringiensis subsp. israelensis is a bacterium that produces crystalline, protein toxins whose activity has been exploited to develop environmentally safe biopesticides that are important in mosquito control. B. t. subsp. israelensis has six genes encoding toxic proteins on a 137 kDa plasmid, including: cry4A, cry4B, cry10A, cry11A (formerly cry4D), cyt1Aa, and cyt2Ba (Ben-Dov et al., 1999). Each component toxin in B. t. subsp. israelensis is mosquitocidal and Cyt2Ba and Cyt1Aa are additionally cytolytic and hemolytic. The toxicity of the native complex of toxins is considerably higher than that of the individual toxins because activity is enhanced by synergism between the component toxins, particularly between Cyt1A and the Cry toxins (Poncet et al., 1995). Interestingly, it was reported that when the cyt1A gene was genetically inactivated, the recombinant strain was as active or nearly as active as native B. t. subsp. israelensis toward susceptible Culex quinquefasciatus, suggesting that this toxin plays little or no significant role in the activity of B. t. subsp. israelensis (Del ecluse et al., 1991). Alternatively, the multi-toxin complex in B. t. subsp. israelensis may have sufficient redundancy that loss of a single toxin has little effect on activity. Although loss of Cyt1A from B. t. subsp. israelensis did not alter toxicity against susceptible mosquito larvae, its absence was discovered to have some important consequences. Mosquito larvae selected with native B. t. subsp. israelensis failed to develop resistance, whereas larvae selected with a strain of B. t. subsp. israelensis lacking the cyt1A gene developed substantial resistance (Georghiou and Wirth, 1997). Further, the resistant mosquitoes remained susceptible to native B. t. subsp. israelensis, despite high levels of resistance and crossresistance to Cry toxins (Wirth and Georghiou, 1997). Because the two bacterial strains differed solely in the presence or absence of the cyt1A gene, it was concluded that this toxin was responsible for suppressing resistance, which was subsequently demonstrated (Wirth et al., 1997). Our understanding of Cyt1A s role in the activity of B. t. subsp. israelensis identifies it as important in toxin synergism, in delaying resistance development, and in suppressing resistance. The contribution of Cyt1A, particularly in synergism and suppressing resistance, suggested that the loss of one component Cry toxin would have less impact on activity against resistant C. quinquefasciatus than would the loss of the Cyt1A toxin. We tested this hypothesis by assaying colonies of C. quinquefasciatus that were selected to moderate or high levels of insecticide resistance toward single or multiple toxins from B. t. subsp. israelensis and comparing the activity of two B. t. subsp. israelensis strains in which either the cry11A gene or the cyt1A gene was disrupted by in vivo recombination (Del ecluse et al., 1991; Poncet et al., 1993). Five toxin preparations, consisting of crystal/spore mixtures of lyophilized powders were assayed. Two preparations were from recombinant strains that produced toxin(s) by expressing cloned gene(s) in acrystalliferous strains of B. thuringiensis, Cry11A (Chang et al., 1992) or Cry4A+Cry4B (Del ecluse et al., 1993). Two bacterial strains were previously developed using homologous recombination to disrupt one component toxin, Deleted CytA, lacking the cyt1A gene (Del ecluse et al., 1991) and Deleted Cry11A, lacking the cry11A gene (Poncet et al., 1993). The fifth strain was native B. t. subsp. israelensis (preparation IPS80, Pasteur Institute, Paris, France). Five colonies of C. quinquefasciatus were evaluated, including a parental reference colony, CqSyn90, and four colonies derived from CqSyn90 that were resistant to one or more B. t. subsp. israelensis toxin(s). The resistant colonies were Cq80 selected with native B. t. subsp. israelensis (IPS 80, resistance ratio at LC95 (RR95), 5.8); Cq4ABD selected with Deleted CytA (RR95, 264); Cq4AB selected with Cry4A and Cry4B (RR95, 101); and Cq11A selected with Cry11A (RR95, >90,000). Bacterial strains were grown on solid or liquid media and crystal/spore powders were prepared using standard methods (Chang et al., 1992; Del ecluse et al., 1991; Poncet et al., 1993). Stock suspensions, in distilled water, were prepared monthly; dilutions were prepared weekly and frozen at )20 C when not in use. Bioassays Journal of Invertebrate Pathology 82 (2003) 133–135 Journal of INVERTEBRATE PATHOLOGY