| Voltage clamp methods, developed to study electrical properties of tissues, were investigated and optimized for use with insect midgut tissues. In optimizing the methodology for use with small tissues, the resistance between electrodes was reduced. The voltage clamp amplifier, ionic strength of the buffer, tissue surface area of the annulus and distance between electrode tips were each investigated and optimized. An optimized voltage clamp chamber was designed and was a major accomplishment of this work. The optimized methods were used to assay Bacillus thuringiensis sensitivity in midgut tissues of fifth instar Lymantria dispar. Two populations, a laboratory population and Manitowoc, WI population, were subjected to the Bt sensitivity assay. The two populations responded in a dose-dependent manner. At the 1400ng dose, the two populations responded differently, in terms of rates of decay and percentage of short-circuit current remaining. Tissues from a New Jersey population were assayed for the control, but were not subjected to the Bt sensitivity assay due to time and equipment constraints. Midgut tissues extracted from larvae collected from a naturally occurring population in Slovakia did not produce short circuit current like the other populations, suggesting variation in physiology between the native and introduced populations. The toxin binding region of the midgut toxin binding protein showed no amino acid sequence variation between populations. Variation in Bt sensitivity between populations may be due to variation in the toxin binding event or other factors. The optimized voltage clamp assay could elucidate physiological phenomena in insect tissues. |
