Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut.

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E(2) (PGE(2)) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE(2) increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

Jaburetox-2Ec: an insecticidal peptide derived from an isoform of urease from the plant Canavalia ensiformis.

Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.

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.