Paul B. Lavrik

Light microscope immunolocation of Bacillus thuringiensis kurstaki δ-endotoxin in the midgut and Malpighian tubules of the tobacco budworm, Heliothis virescens

Light microscope immunofluorescence was used to localize the membrane binding of Bacillus thuringiensis kurstaki 63-kDa delta-endotoxin in Heliothis virescens midgut and Malpighian tubules. Staining was observed along all exposed mucosal (apical microvillar) plasma membranes. Interpretation of the serosal (basal) plasma membrane staining was complicated because the basal lamina also stained. The results suggest that the toxin binds to all exposed plasma membranes without apparent specificity for particular membrane domains.

Effect of Bacillus thuringiensis kurstaki δ-endotoxin on insect Malpighian tubule structure and function

Abstract The effect of Bacillus thuringiensis kurstaki HD-73 δ-endotoxin was determined on the cytoarchitecture and urine transport in isolated Malpighian tubules from larvae of the skipper butterfly Calpodes ethlius. Endotoxins (134 and 64 kDa) were injected into the tubule lumen or applied externally in the bathing medium of Ileac plexus-rectal lead and Yellow region tubules. Mucosal (lumenal) exposure in both tubule regions with the 63 kDa toxin at 16 μg/ml completely inhibited urine secretion and induced massive cytolysis. Serosal (bathing medium) exposure of the 63 kDa toxin in both tubule regions had variable activity and required a 20-fold higher concentration to inhibit fluid secretion and disrupt cell structure. Mucosal exposure to the 134 kDa endotoxin was without effect on cell structure or fluid secretion in both tubule regions confirming its protoxin status. The 63 kDa toxin induced similar cytological alterations in both Ileac plexus-rectal lead and Yellow region Malpighian tubule primary cells. However, mucosal and serosal exposure had different patterns of membrane disorganization, with those membranes closest to the site of application being disrupted first. After 1 hr exposure mucosally, primary cells exhibited variable cytopathological changes with a progression of severity from low to high toxin concentration. Stage 1 (least severe) changes involved enlargement of spaces in the cytoplasm and basal infolds whereas Stage 2 alterations included microvillar disorganization and lysis, mitochondrial alterations, and continued enlargement of cytoplasmic spaces. Stage 3 (most severe) changes included apical and basal membrane lysis, swelling of the rough endoplasmic reticulum and mitochondria, and leakage of cytoplasmic material into the tubule lumen. These results demonstrate that the 63 kDa B. thuringiensis endotoxin affects a fluid-transporting epithelium other than midgut. The similar patterns of activity in two physiologically distinct Malpighian tubule regions by the 63 kDa toxin is most consistent with the idea that the toxin acts by forming pores in cell membranes rather than by poisoning histospecific membrane proteins.