This section describes the process of creating purified midgut BBMV using whole larvae and pupae of Cx. quinquefasciatus and investigates the in-vivo binding of P. fluorescens Migula’s mosquitocidal protein to the mosquito larvae midgut through immunohistochemical methods to determine its primary site of action.
When a susceptible mosquito larva ingests it, the alkaline midgut environment dissolves the crystalline inclusions, releasing the protoxins. The gut proteases then cleave the protoxins into active toxins. The activated toxin fragments bind to specific protein receptors on midgut epithelial cells, leading to membrane insertion and pore formation.
The western blot assay showed the presence of 55kDa and 35kDa bands, proving the binding of mosquitocidal protein to the midgut of treated larvae and pupae of Cx. quinquefasciatus. The immunofluorescence localization research demonstrated that the mosquitocidal protein attaches preferentially to the midgut of larvae and pupae of the mosquito species Cx. quinquefasciatus.
This study confirmed that the mosquitocidal protein binds to the gut regions of the pupae (non-feeding stage) by breaking down the cuticle and overcoming the peritrophic membrane, resulting in effective pupicidal activity. It was discovered that the binding of the protein to the midgut epithelium causes swelling of mitochondrial and endoplasmic reticulum and enlargement of vacuoles, leading to lysis of epithelial cells, midgut perforation and the death of the larvae.
The treated larvae and pupae had lower alkaline phosphatase and amino peptidase activity than the untreated ones, indicating the compromised enzymatic machinery of midgut cells as a result of protein binding to receptor molecules on Brush Border Membrane Vesicles of Cx. quinquefasciatus.
Author(s) Details:
B. Usharani,
Department of Biomedical Genetics, Institute of Basic Medical Sciences, University of Madras, Tamil Nadu, India.
R. Venkateswari,
Department of Medical Microbiology, Institute of Basic Medical Sciences, University of Madras, Tamil Nadu, India.
P. Suganthi,
Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Madras, Tamil Nadu, India.
M. Muthuraj,
State TB Training and Demonstration Centre, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Puducherry, India.
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