MATERIALS AND METHODS

 

The Cyt1Ab1 toxin was obtained from the recombinant strain SPL407-p(CytM) (15). The Cyt1Aa1 recombinant strain was kindly provided by Dr. Federici from California University. The bacteria were grown in Luria-Bertani (LB) suplemented with erythromycin (25 g/ml). A final whole culture of the recombinant strains SPL407-pCytM and Cyt1Aa1 were separated on a discontinuos sucrose gradient (79% [wt/vol] and 67% [wt/vol]) (16). The pure crystals were collected, washed and checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE 10%) according to Laemmli (8). The solubilization of Cyt1Ab1 crystals was performed at different pH values (Table 1), the soluble protein concentration was determined by the Bio-Rad technique using bovine serum albumin as standard.

The gut extract was prepared from third instar C. quinquefasciatus larvae, after dissection they were collected in MET buffer (Mannitol 300 mM, EDTA 5 mM, Tris 20 mM pH 7.2), disrupted and centrifuged. Then the supernatant with protease activities was recovered, and protein concentration was determinated.

The Cyt1Ab1 toxin inclusions were in vitro treated with trypsin, chymotrypsin and C. quinquefasciatus gut extracts in a 1:10 ratio (w/w, enzyme:toxin) in different buffer solutions (Table 1). The digested protein was examined by SDS-PAGE 10%, and analyzed by the NIH program v. 1.61 (14). The processing of the Cyt1Ab1 toxin inclusions in the time was achieved with a 1:2.5 ratio (w/w, enzyme/ toxin) in carbonate buffer pH 11, and aliquots of 10 l were collected at several time intervals (1, 5, 10, 30 min, 1, 2, 3, 14, and 24 h). Samples were then examined by SDS-PAGE 10% as above. The in vivo processing of the toxin was performed using third instar C. quinquefasciatus larvae. After intoxication the entire larvae were washed extensively in distilled water, the guts were dissected, separated by SDS-PAGE 10%, transferred to a nitrocellulose membrane and probed using an anti-Cyt1Ab1 polyclonal antibody.

The in vitro hemolysis assays were carried out as described by Thiéry et al. (15), with modifications. The Cyt1Ab1 toxin was solubilized and treated with proteases and C. quinquefasciatus gut extracts. Sheep red blood cells (2.25%) were incubated with dilutions of the activated toxin. The mosquitocidal activity of Cyt1Ab1 and Cyt1Aa1 toxin crystal inclusions were tested against third instar C. quinquefasciatus larvae according to Thiéry et al. (15). The half hemolytic dose (HD50) and the half lethal concentration (LC50) were calculated by Probit analysis (13).