Stefan Jansens

Immunocytochemical localization of Bacillus thuringiensis insecticidal crystal proteins in intoxicated insects

The damage of different insecticidal crystal proteins (ICPs) from Bacillus thuringiensis to the midgut of Manduca sexta, Plutella xylostella, and Leptinotarsa decemlineata larvae was determined by light microscopic observations. During the intoxication process, the distribution of the different ICPs in the larval body was monitored with specific antibodies. In lepidopteran and in coleopteran insects, histopathological changes include disruption of the brush border, vacuolization of the cytoplasm, hypertrophy of the epithelial cells, and disintegration of the cell. After ingestion by the insect larvae, the ICPs rapidly accumulate in the peritrophic membrane. However, the binding to the peritrophic membrane does not correlate with toxicity, since the coleopteran-specific toxin (CryIIIA) is also retained in the peritrophic membrane of lepidopteran insects, while the lepidopteran-specific toxin CryIA(b) binds to the peritrophic membrane of the Colorado potato beetle larvae. In contrast, ICPs bind to the microvilli of the midgut epithelial cells of susceptible insects only, confirming a correlation between toxicity and binding to the brush border membrane. In the lepidopteran larvae, the lepidopteran-specific toxic ICPs initially accumulate at the apical microvilli of the epithelial cells in the anterior part of the midgut. In the Colorado potato beetle larvae, CryIIIA is primarily retained by the microvilli of the epithelial cells from the posterior part of the midgut. During the intoxication process, internalization of the ICPs into midgut epithelial cells is not detected, even several hours after toxin ingestion. Apparently, the ICPs are retained within the gut since they were not detected in other organs such as the Malpighian tubules.

Immunocytochemical analysis of specific binding of Bacillus thuringiensis insecticidal crystal proteins to lepidopteran and coleopteran mudgut membranes

Abstract The in vitro binding of four insecticidal crystal proteins (ICPs) from Bacillus thuringiensis to midgut tissue sections of two lepidopteran ( Manduca sexta and Plutella xylostella ) and one coleopteran ( Leptinotarsa decemlineata ) species was immunocytochemically analyzed. Monoclonal antibodies, highly specific against the different ICPs, were used. Light microscopy observations showed that the crystal proteins accumulated at the exposed peritrophic membrane of all lepidopteran insects and at the apical microvilli of sensitive lepidopteran insects. No accumulation was observed at the microvilli present in the goblet cell cavity or at the Malpighian tubules. For all lepidopteran insects the binding of the different ICPs to the apical microvilli of the midgut epithelium appears to be highly specific: only the ICPs which were toxic bound to the apical microvilli of midgut tissue sections. As an exception, the nontoxic CryIB bound to the apical microvilli of M. sexta . The binding of ICPs to the peritrophic membrane was not correlated to toxicity. For the first time binding of an ICP to a coleopteran insect larva has been shown. Only the coleopteran-specific toxin CryIIIA bound to the brushborder and to the peritrophic membrane. The binding is preferential to the apical microvilli in the posterior part of the midgut. These results suggest that the mechanism of ICP toxicity in coleopteran insects may be slightly different from that in Lepidopteran and/or Diptera. The present procedure is sensitive and rapid, can be applied to individual insects, and as such may prove very useful for analyzing the binding of different ICPs to small insects.

Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki.

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.