The potential mosquitocidal activity of cry4A toxic region crystal protein gene from local isolates of Bacillus thuringiensis against Aedes aegypti

Abstract

The present study was conducted to isolate Bacillus thuringiensis (B.t.) strains from different ecological regions of Pakistan and determined the mosquitocidal activity of cry4A crystal protein gene against the dipteran insects. Out of 25 B.t. isolates, 5 isolates were selected on the basis of toxicity and 4 were identified as Bacillus thuringiensis GCU-DAB-TK-04 (MN922746), GCU-DAB-TK-06 (MT032397), GCU-DAB-TK-09 (MN922747), and GCU-DAB-TK-13 (MT032398). GCU-DAB-TK-04 was found to be the most toxic strain against the 3rd instar larvae of Aedes aegypti. The presence of Cry 4A protein was confirmed by the amplification of cry4A gene (accession number MT001910) from GCU-DAB-TK-04. The highest spores toxicity was shown by GCU-DAB-TK-04 (LC50 = 104 μg/ml) while LC50 values of other strains were GCU-DAB-TK-13 (LC50 = 602 ± 0.43 μg/ml) GCU-DAB-TK-06 (LC50 = 812 ± 0.63 μg/ml), GCU-DAB-TK-12 (LC50 = 1230 ± 1.14 μg/ml), and GCU-DAB-TK-09 (LC50 = 7585 ± 1.17 μg/ml). Similarly, the highest toxicity of total cell protein was also shown by GCU-DAB-TK-04 (LC50=676 ± 0.53 μg/ml while LC50 values of other strains were GCU-DAB-TK-13 (LC50 = 724 ± 1.12 μg/ml), GCU-DAB-TK-06 (LC50 = 741 ± 0.64 μg/ml), GCU-DAB-TK-12 (LC50 = 912 ± 0.65 μg/ml), and GCU-DAB-TK-09 (LC50 = 1621 ± 1.13 μg/ml). The order of toxicity of B.t. strains was GCU-DAB-TK-04 > GCU-DAB-TK-13 > GCU-DAB-TK-06 > GCU-DAB-TK-12 > GCU-DAB-TK-09. Protein analysis showed that 130 kDa (probably Cry4A, B), 70 kDa (Cry4C, D) and 20 kDa (Cyt enhancer protein of Cry4) proteins were present in all B.t. strains. These B.t. strains have found great potential to grow into bio-insecticidal formulation for the eco-friendly control of mosquitoes.