Highly pathogenic strains of Bacillus sphaericus produce the mosquitocidal Bin proteins, but resistance to this toxin can be produced under laboratory and field conditions. Analysis of strains able to overcome this resistance revealed the presence of a previously undescribed type of two-component toxin. One subunit, Cry48Aa1, is related to the 3-domain crystal toxins of Bacillus thuringiensis. Uniquely for this type of protein, insect toxicity is only achieved in the presence of a second, accessory protein, Cry49Aa1. This protein is itself related to both the binary toxin of B. sphaericus and to Cry35 and Cry36 of B. thuringiensis, none of which require interaction with Cry48Aa1-like proteins for their activity. The necessity for both Cry48Aa1 and Cry49Aa1 components for pathogenicity, therefore, indicates an unprecedented interaction to generate toxicity. Despite high potency for purified Cry48Aa1/Cry49Aa1 proteins (LC50 for third instar Culex quinquefasciatus larvae: 15.9 ng/ml and 6.3 ng/ml respectively), bacteria producing them show suboptimal mosquitocidal activity due to low-level Cry48Aa1 production. This new toxin combination may indicate a fortuitous combination of members of the gene families that encode 3-domain Cry toxins and Binary-like toxins, permitting the "mix-and-match" evolution of a new component in the mosquitocidal armoury.

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The cotton boll weevil (Anthonomus grandis) is the major cotton pest in the Americas. One of the alternatives for its control is the utilization of Bacillus thuringiensis (Bt), an entomopathogenic bacterium characterized by its production of insecticidal crystal proteins. Embrapa Genetic Resources and Biotechnology has a collection of Bacilli in which diVerent isolates of Bt are stored. A method for rearing and maintenance of Anthonomus grandis on artiWcial diet in the laboratory was developed, and a robust larval bioassay protocol was established for the selection of B. thuringiensis isolates toxic to boll weevil. After preliminary bioassays performed with 215 isolates, 5 were selected that demonstrated a good level of toxicity and these were analyzed in more detail. The most toxic were S601 and S1806 presenting LC 50 (lethal concentration to kill 50% if the larvae) of 0.14 mg/ml and 0.30 mg/ml, respectively. S601 showed an LC 50 value that was half that of the standard B. thuringiensis subspecies tenebrionis and S1806 demonstrated similar values of LC 50 to the standard. S601 contained the cry1B gene and S1806 had the cry4A, cry4B, cry10, cry11, cyt1 and cyt2 genes like B. thuringiensis subspecies israelensis.

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