Ex vivo Cytotoxicity of the Bacillus thuringiensis Cry4B δ-Endotoxin to Isolated Midguts of Aedes aegypti Larvae

• Published in: BMB reports Vol. 36; no. 3; pp. 294 - 298
• Main Authors: Sahawat Barusrux, Issara Sramala, Gerd Katzenmeier, Ahnond Bunyaratvej, Sakol Panyim, Chanan Angsuthanasombat
• Format: Journal Article
• Language: Korean
• Published: 생화학분자생물학회 30.05.2003
• Subjects: Bacillus thuringiensis; Cell viability assay; Ex vivo toxicity; Mosquito-larval midgut; Toxic potency; δ-Endotoxin
• ISSN: 1976-6696

The pathological effect of the Bacillus thuringiensis Cry δ-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the cioreduced formazan product at 490 mm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protein. however, the trypsin-treated products of the non-hioactive Cry4B mutant (R158A) and the lepodopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.