Electron probe x-ray microanalysis of the effects of Bacillus thuringiensis var Kurstaki crystal protein insecticide on ions in an electrogenic k+-Transporting epithelium of the larval midgut in the lepidopteran, Manduca sexta, in vitro

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Abstract
An alkaline hydrolysate of Bacillus thuringiensis var kurstaki HD 1 (Btk) parasporal crystals was administered at 25 μgml-1 (f.c.) to isolated, short-circuited, midguts of tobacco homworm (Manduca sexta) larvae. The short-circuit current (s.c.c.), a precise measure of K+ active transport, was inhibited by 78% in 10 min in Btft-treated midguts as compared to controls. The elemental concentrations of K, together with Na, Mg, P, S, Cl and Ca, as well as the water content, were determined by electron probe X-ray microanalysis (EPXMA) in the muscle cells, columnar cells and goblet cells, as well as in the extracellular goblet cavity and the bathing media. The average K concentration in the goblet cell cavity was 129mmol/kg wet wt in control midguts but only 37 mmol/kg wet wt in fitA-treated midguts. The elemental concentrations, including that of K, in other cell compartments were much less affected by Btk, but a rise in total cell calcium is suggested. It has been previously suggested that in vivo Btk acts specifically on limited regions of the apical membrane of the midgut epithelial cells. The simplest interpretation of the EPXMA results would be that initially Btk interacts specifically with the goblet cell apical membrane, which bounds the goblet cavity and contains the K+ pump responsible for the s.c.c. and high transepithelial potential difference (p.d.). Such interaction results in a rapid disruption of K+ transport across the goblet cell apical membrane, leading to dissipation of the K+ gradient and loss of p.d. The histopathological changes previously reported by other workers would then be a consequence of K+ pump inhibition causing changes in the intracellular pH, Ca2+ etc. Some possible molecular bases for these specific interactions between Btk and cell membrane are discussed.