Improving the Insecticidal Activity against Resistant Culex quinquefasciatus Mosquitoes by Expression of Chitinase Gene chiAC in Bacillus sphaericus

Abstract

Expression of a chitinase gene, chiAC, from Bacillus thuringiensis in B. sphaericus 2297 using the binary toxin promoter yielded a recombinant strain that was 4,297-fold more toxic than strain 2297 against resistant Culex quinquefasciatus. These results show that this chitinase can synergize the toxicity of the binary toxin against mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus.Insects of dipteran species such as Culex spp., Anopheles spp., and Aedes spp. are responsible for the transmission of many infectious disease agents. Directing an attack against these mosquitoes is recognized as one of the more effective approaches for eradicating the threat from infectious diseases (4). As a gram-positive, spore-forming, aerobic, entomopathogenic bacterium, Bacillus sphaericus has been successfully used for mosquito control in the last decade. Its activity against target mosquito larvae is mainly due to the crystal toxin, commonly referred to as the binary toxin, as it consists of equimolar amounts of two proteins of 51 and 42 kDa. (1). However, high-level resistance against B. sphaericus binary toxin after intensive treatment has developed in mosquitoes (6), with resistance ratios ranging from 35-to 150,000-fold in the laboratory (5, 8) and from 10-to 100,000-fold in the field (15). Experiments testing binary toxin binding to the mosquito larval midgut in vitro have demonstrated that resistance of mosquitoes to B. sphaericus binary toxin has occurred mainly because of elimination of the toxin-binding site on the midgut brush border membrane fraction (1, 6). The appearance of high-level of resistance in mosquitoes is a threat to the future application of B. sphaericus as a mosquito control agent.On the basis of the sequence of the chi gene from B. thuringiensis subsp. israelensis (GenBank accession no. AF526379), two pairs of primers, chiAC-1-chiAC-3 and chiAC-2-chiAC-3, were designed for amplifying the chiAC open reading frame by PCR with different enzyme digestion sites introduced from strain T04A001 (3) (Table 1) as described elsewhere (17). After digestion with EcoRI and HindIII, the PCR fragment amplified with primers chiAC-1 and chiAC-3 was ligated with EcoRI/HindIII-digested plasmid pET28a and the resulting plasmid was transformed into Escherichia coli BL21. The recombinant E-pETC21 was selected on LB agar supplemented with kanamycin (50 g/ml). The 70-kDa ChiAC fusion protein was purified for preparation of rabbit anti-ChiAC with a His-Bind Resin chromatography kit by following the procedure provided by the manufacturer (Novagen) (9).For the expression of chiAC in B. sphaericus, a binary toxin promoter was amplified as described elsewhere (14) with primers pbinary-1 and pbinary-2. The amplified binary toxin promoter (ϳ0.5 kb) was digested with BamHI and SphI and then introduced into BamHI/SphI-digested vector pBU4, resulting in recombinant plasmid pBb. The open reading frame of ChiAC, obtained by PCR with primers chiAC-2 and chiAC-3, was inserted into pBb at the SphI and HindII...

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

“…The latter explanations may hold true for the native Bt 55 strain described in this study, which had enhanced mosquitocidal activity, a similar cry and cyt gene profile as that of Bti -H14, but also possessed the Chi gene. The chitinase gene has been shown to elevate the mosquitocidal effect by perforating the peritrophic membrane, thereby increasing the accessibility of the Cry and Cyt toxins to receptors on the epithelial membrane [7, 47]. Abdullah et al [48] further experimentally demonstrated that transferring the chitinase gene from B. subtilis to a Bt strain improved its insecticidal bioactivity.…”

Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus

“…quinquefasciatus (Cai et al, 2007). These results show that chitinase can synergize the toxicity of Bin and thus may be useful in managing larval resistance to B. sphaericus .…”

A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects

“…Some virulence factors enhance the insecticidal activity of the Cry protein by destroying the epithelial cells of the insect midgut. For example, an enhancing‐like protein Bel1 can enhance the toxicity of Cry1Ac by degrading the peritrophic matrix (PM) and intestinal mucin (IIM) of Trichoplusia ni and Helicoverpa armigera larvae midguts (Fang et al ., ); chitinases produced by B. thuringiensis can increase the activity of Cry proteins against different insect targets (Tantimavanich et al ., ; Cai et al ., ); an M4 metalloproteinase Bmp1 is toxic to Caenorhabditis elegans and exhibits intestinal tissue degradation activity to enhance the toxicity of Cry5Ba against C. elegans (Luo et al ., ). Meanwhile, some virulence factors may facilitate better germination and colonization of B. thuringiensis in insects.…”