Efeito entomotóxico da urease de canavalia ensiformis (jbu) e do peptídeo tóxico jaburetox (jbtx) sobre a junção neuromuscular de baratas da espécie nauphoeta cinerea

Abstract

The growth of world population has generated a necessity of an increase in agricultural productivity using ecological pesticides. For these reasons, the natural compounds have been studied in order to develop safer products for consumers and to the environment. Plants have a range of biological and physicochemical properties that provide several possibilities of application, varying from the medical application to products for controlling the agricultural pests. Ureases are enzymes that are involved in plant defense mechanisms, showing entomotoxic properties, which are unrelated to its enzymatic activity. In addition, there are several isoforms of ureases, but this work covers only those related to the Jack Bean urease (JBU) isoform and its recombinant toxic peptide, Jaburetox. Therefore, the aim of this study was to investigate the entomotoxic effect of the Canavalia ensiformis urease (JBU) and its recombinant toxic peptide, Jaburetox (Jbtx) on the neuromuscular junction of Nauphoeta cinerea cockroach. In order to accomplish this, in vivo neuromuscular preparations of adult cockroaches were used together with in vitro assays of acetylcholinesterase activity, of the cockroach brain homogenate. The heterologous expression of Jaburetox peptide was carried out in Escherichia coli, and its purification was made by a nickel column chromatography. Thereby, our results show that JBU significantly inhibits the acetylcholinesterase (AChE) activity. When the cockroaches were treated with JBU (1.5; 3 and 6μg/g), there was an inhibition of 21.2 ±3%, 33±2% and 60±3% of AChE activity, respectively. The electromyographic assay shows that JBU induced a time-dependent neuromuscular blockage. When the 1.5 and 3μg/g doses were assayed, there was a blockage of 30±10% and 15±4% of twitch tension, respectively, after 120min of recordings. Moreover, the administration of the higher dose JBU (6μg/g) increased the neuromuscular inhibition (65±8%), after 120min of recording. Nevertheless, the pretreatment of the animals with dexamethasone (40μg/g) partially inhibited (~45%) the neuromuscular blocking effect of JBU (6μg/g). Furthermore, the electromyographic assay with Jbtx also induced a time-dependent neuromuscular blockage. Thus, when the 2.5; 5 and 10μg/g doses were assayed, there was a blockage of 76±16%, 68±10% and 71±8% on twitch tension, after 120min of recording. Even though, when the higher dose (15μg/g) was assayed, there was no significative inhibition of the neuromuscular inhibition (22±12%). Taken together, the results indicate that JBU significantly inhibits the AChE activity in insects. Additionally, JBU (1.5 and 6μg/g) induced a progressive and significant inhibition of the neuromuscular twitches of N. cinerea cockroaches. However, the pretreatment with dexamethasone decreases the JBU effect. Furthermore, Jbtx in lower doses (2.5, 5 and 10mg/g) also induced neuromuscular blockage of N. cinerea cockroach. Finally, success was achieved in the process of expression and purification of the peptide Jaburetox in E. coli, yielding in the end of the process, approximately 330μg/mL of the peptide. In conclusion, this work demonstrated the viability of Canavalia ensiformis urease and its peptide Jaburetox as natural pesticides, which induces the peripheral neurotoxicity in target insects.