Midgut Binding Activity of Mosquitocidal Extracellular Protein of Pseudomonas fluorescens Strain

This chapter aimed to describe the preparation of purified midgut BBMV using whole larvae and pupae of Cx. quinquefasciatus as a starting material and investigated the in-vivo binding of the mosquitocidal protein of P. fluorescens Migula B426 to the mosquito larvae midgut through immunohistochemical methods to determine this protein's primary site of action.

Upon ingestion by a susceptible mosquito larva, the alkaline midgut environment promotes solubilization of crystalline inclusions releasing the protoxins. Subsequent cleavage by gut proteases results in formation of active toxins. The activated toxin fragments then bind to specific protein receptors on midgut epithelial cells, leading to membrane insertion and pore formation. The presence of 55kDa and 35kDa bands in western blot assay revealed the binding of mosquitocidal protein to the midgut of treated larvae and pupae of Cx. quinquefascistus. The mosquitocidal protein attaches preferentially to the midgut of larvae and pupae of the mosquito species Cx. quinquefasciatus, according to the immunofluorescence localization research. The present investigation confirmed that the binding of mosquitocidal protein to the gut regions of the pupae (non-feeding stage) by degrading the cuticle and overcoming the peritrophic membrane and thus found to be effective pupicidal activity. It has been reported that the binding of the protein to the midgut epithelium causes swelling of mitochondrial and endoplasmic reticulum and enlargement of vacuoles, followed by lysis of epithelial cells, midgut perforation and the death of the larvae. It was discovered that the treated larvae and pupae had lower alkaline phosphatase and amino peptidase activity than the untreated larvae and pupae. The findings showed that the enzymatic machinery of midgut cells was compromised and the specific activity of the enzymes was decreased as a result of protein binding to receptor molecules on Brush Border Membrane Vesicles of Cx. quinquefasciatus.