The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from C u k x pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Bacillus sphaericus is a Gram-positive spore-forming bacterium and is one of the most promising agents for the control of mosquitoes. Some strains produce a very high larvicidal activity, especially for mosquito species belonging to the Culex and Anopheles genera, while they are almost inactive on Aedes species. The insecticidal activity is directly related to the synthesis of parasporal protein crystals during sporulation (Davidson, 1983;Payne and Davidson, 1984;Kalfon et al., 1984;Baumann et al., 1985). These crystals contain two major polypeptides of 41.9 kDa and 51.4 kDa, as deduced from DNA sequence data (Hindley and Berry, 1987;Baumann et al., 1988;Arapinis et al., 1988; for complete review see Baumann et al., 1991). Bacillus subtilis or Escherichia coli expressing either of the genes encoding these two proteins are poorly toxic or even non-toxic for mosquito larvae (Baumann et al., 1988;de la Torre et al., 1989;Broadwell et al., 1990). However, transformants expressing both genes are fully toxic, indicating that the protein crystal of B. sphaericus acts as a binary toxin (Broadwell et al., 1990;Baumann and Baumann, 1991).Little is known about the mode of action of these toxins, except that they appear to exert cytopathological effects (Davidson, 1981;Karch and Coz, 1983; Charles, 1987) by binding to susceptible mosquito larval midgut cells (Davidson et al., 1987;Davidson, 1988Davidson, ,1989. In addition, both proteins bind to the gastric caecae and posterior midgut of Culex larvae when administered together (Davidson et al., 1990).In this report, we demonstrate that B. sphaericus binary toxin specifically binds to susceptible mosquito larval midgut membranes, and we measure the affinity of the toxin for its putative membrane receptor.
MATERIALS AND METHODS
Insects and toxicity assaysLarvae of Culexpipiens ssp. pipiens L. (Montpellier strain) and Aedes aegypti L. (Bora-Bora strain) were reared on cat biscuits at 28 "C. Adult populations were maintained on honey solution, at 2 8 T , in 80% relative humidity with a 16 hi8 h photoperiod. Females were fed on guinea pigs.Toxicity assays were performed on early fourth-instar larvae, in 5-cm diameter Petri dishes containing 10 ml bacterial or crystal suspension, or soluble protein dilutions in demineralized water. Dead larvae were counted after 24 h and 48 h, and lethal concentrations killing 50% of the population (LC,,) evaluated by probit analysis (Finney, 1971).
Brush-border membrane isolationBrush-border membrane fractions (BBMF) were prepared from midguts of C. pipiens and A . aegypti larvae following methods modified from those of Biber et al. (1981; described in Wolfersberger et al., 1987) and Houk et al. (1986). Fourthi...