Insecticidal effects of canatoxin on the cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae).

Canatoxin (CNTX) is a variant form of urease isolated from Canavalia ensiformis (Leguminosaea) seeds. A possible role in the plant defense was proposed for CNTX, due to its toxicity upon feeding to the beetle Callosobruchus maculatus, and the hematophagous bug, Rhodnius prolixus. The toxic effect is caused by a canatoxin-derived peptide ( approximately 10kDa) formed by insect cathepsin-like digestive enzymes. In order to evaluate their potential as bioinsecticides, the effects of CNTX and its peptide were evaluated on a phytophagous hemipteran insect Dysdercus peruvianus, a pest of cotton culture. For the bioassays, the insects fed on gelatin capsules containing powdered cotton seeds, mixed with the freeze-dried protein and other test materials and were observed for survival rate, weight gain and molting. Ingestion of canatoxin, or a recombinant 10kDa peptide derived from it, severely affected young forms of the insects, delaying their development or leading to their death. In contrast, adults were insensitive to diets containing higher concentrations of canatoxin. Cathepsin-like proteinases predominated and showed distinct pattern of enzymatic activities in midguts homogenates according to the developmental stage of the insect, a fact which may explain the different susceptibility of nymphs as compared to adult D. peruvianus. The data presented confirm the potential use of canatoxin-like proteins and derived peptides as bioinsecticides.

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes.

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.