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Title: REGENERATION OF CULTURED MIDGUT CELLS AFTER EXPOSURE TO SUBLETHAL DOSES OF TOXIN FROM TWO STRAINS OF BACILLUS THURINGIENSIS

Author
item LOEB, MARCIA
item MARTIN, PHYLLIS
item HAKIM, RAZIEL - HOWARD UNIV. WASH. DC
item GOTO, SHINTARO - KOBE UNIV., KOBE, JAPAN
item TAKEDA, MAKIO - KOBE UNIV., KOBE, JAPAN

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Heliothis virescens is a severe pest to crops of cotton, tobacco, corn and other vegetables. It has recently been possible to control this pest by use of toxin from the bacterium, Bacillus thuringiensis (Bt). Once injested, the toxin breaks down to an active form in the caterpillar midgut and attacks mature gut cells, causing them to swell and disintegrate. However, insufficient doses of toxin do not kill insects, and many strains of Heliothis have become resistant to the toxin. Therefore, it is important to find out how the midgut of Heliothis copes with lower doses of Bt toxin. We study midgut cells of Heliothis grown in tissue culture. In this study we found that the toxin causes many mature cells to break apart, but at the same time, induces the stem cells to multiply rapidly; they then differentiate to mature cells rapidly. In an intact insect, this would result in replacement of dying mature cells by newly differentiated cells. When the toxin is washed out of the system, the population of cells returns to its original ratio of cells and becomes normalized. This explains why lower doses of Bt toxin do not "work" at all, and presents the necessity of depleting the cells of the replacement system with high enough doses of Bt toxin to cause rapid loss of so many midgut cells that replacement cannot be effected before the insect dies. This information will be useful to other scientists studying Bt as well as to applicators of the toxin for pest control.

Technical Abstract: Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective in eliminating columnar and goblet cells although, at the doses used, some mature, stem and differentiating cells remained. After 2 days of exposure to 0.8 pg/ul AA 1-9 or0.06 pg/ul HD-73, the numbers of columnar and goblet cells declined to approximately 40% of controls while stem and differentiating cells increased to 150 to 200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid turnover in vitro in times of duress and recovery. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells appeared to be induced the cultured midget cells themselves. Controls and cells treated with toxin from Bt strain AA1-9 were stained using a polyclonal antibody to Lepidopteran midget differentiation factor 1(MDF1). After incubation with Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis during increased stem cell differentiation. The response of cultured midget cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.