Abstract
Mosquitoes vector many infectious diseases and pose a serious threat to public health in developing countries. A wide array of synthetic insecticides is employed against these vectors. As the mosquitoes have got resistance to most of these chemical insecticides, there is a dire need to explore alternative control strategies such as entomopathogens. This laboratory study was aimed at isolating, purifying and evaluating the insecticidal proteins from indigenous strains of soil-inhabiting pathogenic bacteria against the larvae of southern house mosquito (Culex quinquefasciatus) and to identify the larval midgut receptor of bacterial toxin. One hundred bacterial isolates were isolated and screened. Only two isolates (AA-11 and AA-19) exhibited considerable larval mortality (> 80%) and were further identified by sequencing and blasting their 16 S rDNA gene fragments. Isolate AA-11 was identified as Pseudomonas sp. with 96% similarity, while AA-19 as Lysinibacillus sphaericus with 98% similarity. Microscopic analysis and gram staining of AA-19 confirmed its rod-shape structure and gram positive nature, respectively. It is likely that the bacterial isolate AA-19 was L. sphaericus. Furthermore, protein purification of this bacterial isolate revealed two proteins of approximately 51 and 43 kDa which reduced to 43 and 39 kDa respectively after digestion with trypsin indicating that these were Bin B and Bin A toxin proteins respectively of L. sphaericus. Results of toxicity bioassay revealed that the purified and activated toxin was highly toxic to C. quinquefasciatus larvae with a LC50 value of 191 ppb. Pull-down assays demonstrated that one protein of 66.5 kDa was pulled down by CNBr-beads conjugated with insecticidal proteins (Bin B and A). The pulled down protein was considered as C. quinquefasciatus maltase 1 (Cqm1), a receptor of Bin toxin in the midgut of mosquito larvae. After binding with its receptor, it is probable that Bin toxin induced vaculation, pore formation, autophagy and apoptosis in the midgut cells of C. quinquefasciatus larvae to cause septicemia. Conclusively, the purified and activated insecticidal protein (Bin toxin) can be a key part of integrated vector management and has potential to be developed as commercial microbial formulation against mosquito larvae with a low risk to environment and non-target insects. The present study can be helpful to control the vectors of important diseases by understanding the interaction and binding of toxin and receptor.
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Acknowledgements
Authors are thankful to Dr. Salman Ahmed (Department of Plant Pathology, College of Agriculture, University of Sargodha, Sargodha, Pakistan) for the technical assistance during the study.
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This research work was financially supported by the Higher Education Commission of Pakistan (HEC) under its NRPU Program (Project No. NRPU-11326).
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MAR and SA conceived the idea and designed the study. SI, AA and ZA performed experimentation. MAR and SI analyzed the data and prepared results. MAR and MZM wrote the first draft of manuscript. MZM and SA technically proofread the manuscript. MAR and MS supervised the research work. MA and MAR provided technical and financial assistance to the study. All authors have read and approved the final version of the manuscript.
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Iftikhar, S., Riaz, M.A., Majeed, M.Z. et al. Isolation, characterization and larvicidal potential of indigenous soil inhabiting bacteria against larvae of southern house mosquito (Culex quinquefasciatus Say). Int J Trop Insect Sci 43, 781–791 (2023). https://doi.org/10.1007/s42690-023-00992-x
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DOI: https://doi.org/10.1007/s42690-023-00992-x