[The synergism between Mtx1 from Bacillus sphaericus and Cyt1 Aa from Bacillus thuringiensis to Culex quinquefasciatus].

Mosquitocidal toxin 1 (Mtx1) was synthesized during vegetative phase of Bacillus sphaericus and it had been proved to have higher activity to Aedes spp. larvae and Binary toxin (Bin) resistance Culex larvae. The truncated 97 kDa Mtx1 with a deletion of the signal peptide and the Cyt1 Aa crystal protein, a 27.3 kDa delta-endotoxin from Bacillus thuringiensis subsp. israelensis (Bti), were purified from Escherichia coli and B. thuringiensis recombinant strains respectively. Both purified toxins had high toxicity against Culex quinquefasciatus larvae. Bioassay result revealed the purified Mtx1 toxin had high toxicity against the target mosquito larvae, with LCso of 45.2 ng/mL. However, the mixture of Mtx1 and Cytl Aa exhibited higher toxicity against the mosquito larvae, with a lowest LC50 value of 20.19 ng/mL at the ratio of 1:3. (Mtx1:Cyt1 Aa). The calculated synergistic factor of different mixtures suggested a strong synergistic effect between Cyt1 Aa toxin and Mtx1. Furthermore, the presence of Cyt1Aa in the mixture could induce early larval mortality, enhancing the activity of Mtx1 to the target mosquito larvae. The synergistic effect of Cyt1 Aa on mortality of Mtx1 to mosquito larvae might be caused by the damage of the larval midgut-hemocoel barrier induced by the activated CytlAa toxin, which enhanced the specific pathogenesis of Mtx1 on mosquito larvae. It is suggested that the co-application of Mtx1 and Cyt1 Aa in future will be integrated for mosquito management.

[Domain swapping of Cry1Aa and Cry1Ca from Bacillus thuringiensis influence crystal formation and toxicity].

Bacillus thuringiensis has been successfully used for agricultural pest and medical insect control with its significant benefits based on environmental and safety considerations. However, the deficiency of this pesticide, such as limited spectrum of insecticidal activity, low toxicity to the targets and the inducement of insect resistance, results in the urgent need to exploit new resources of B. thuringiensis or to modify known strains by genetic engineering. In this study, six recombinant Bacillus thuringiensis BT-ACC, BT-AAC, BT-ACA, BT-CAA, BT-CCA and BT-CAC were constructed through the domain swapping between crystal protein CrylAa and CrylCa. SDS-PAGE and Western blot revealed that only the recombinant BT-CAA and BT-CCA produced a 135kDa chimeric protein CrylCAA and CrylCCA respectively, but the production level was lower than the native protein CrylAa and CrylCa. These chimeric crystal proteins could be activated by trypsin, giving a 65kDa protease-resistant core toxin as the native crystal proteins CrylAa and CrylCa. Electron microscopy study indicated that the two chimeric proteins could be produced and accumulated as spherical or granules crystals during sporulation of BT-CAA and BT-CCA, whereas native CrylAa and CrylCa were accumulated as bipyramidal crystals. Unexpectedly, the toxicity of purified chimeric crystal proteins Cryl CAA and Cryl CCA was 3 - 5 times lower to Spodoptera exigua, and 190 - 260 times lower to Helicoverpa armigera than that of native CrylAa and CrylCa. These data suggested that the domain swapping of different crystal proteins might influence the formation and toxicity to targets of chemeric crystal proteins.

Single nucleotide deletion of cqm1 gene results in the development of resistance to Bacillus sphaericus in Culex quinquefasciatus.

The entomopathogen Bacillus sphaericus is one of the most effective biolarvicides used to control the Culex species of mosquito. The appearance of resistance in mosquitoes to this bacterium, however, remains a threat to its continuous use in integrated mosquito control programs. Previous work showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase present in the larval midgut microvilli. In this work, we studied the molecular basis of the resistance developed by Culex quinquefasciatus to B. sphaericus C3-41. The cqm1 genes were cloned from susceptible (CqSL) and resistant (CqRL/C3-41) colonies, respectively. The sequence of the cDNA and genomic DNA derived from CqRL/C3-41 colony differed from that of CqSL one by a one-nucleotide deletion which resulted in a premature stop codon, leading to production of a truncated protein. Recombinant Cqm1S from the CqSL colony expressed in Escherichia coli specifically bound to the Bin toxin and had α-glucosidase activity, whereas the Cqm1R from the CqRL/C3-41 colony, with a deletion of three quarters of the receptor's C-terminal lost its α-glucosidase activity and could not bind to the binary toxin. Immunoblotting experiments showed that Cqm1 was undetectable in CqRL/C3-41 larvae, although the gene was correctly transcribed. Thus, the cqm1R represents a new allele in C. quinquefasciatus that confers resistance to B. sphaericus.