Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000-and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000-and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.

Aims: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. Methods and Results: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirtyeight strains produced different levels of chitinase at pH 7AE0, and so did 17 strains at pH 10AE0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml )1 in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2AE35-fold. Conclusion: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. Significance and Impact of the Study: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide. INTRODUCTIONChitin, the b-(1,4)-linked homopolysaccharide of N-acetylglucosamine, is the second most abundant polysaccharide in nature. It occurs in insects as a major component of the cuticle and of the peritrophic membrane, a protective sleeve lining the gut of many insects (Kramer and Koga 1986;Cabib 1987). Chitinase, which is defined as an enzyme (EC 3.2.1.14) with a specific hydrolytic activity directed against the homopolymer chitin, might play a key role in virulence of some pathogens that infect insects via the peritrophic membrane. Enzymatic cleavage occurs randomly at internal locations over the entire length of the chitin microfibril, leading to the impairment of the insect midgut (Kramer and Koga 1986;Hawtin et al. 1997;Kramer et al. 1997). Hypothetically, chitinase causes perforations in the gut peritrophic membrane, which facilitate entry of the pathogens into the haemocoel of susceptible insects (Brandt et al. 1978).The Gram-positive bacterium Bacillus thuringiensis is an important industrial organism and is used worldwide for pest control in agriculture, horticulture and silviculture, as well as for the control of disease-related insect vectors. Its specific activities against a variety of insects are attributed to the parasporal crystals, which are produced and assembled during sporulation. After ingestion by target insects (usually the larvae), the crystals are solubilized and converted into active toxins by proteases in the larval midgut. The active toxins bind to specific receptors on the surfaces of the midgut epithelium cells, and insert into the me...

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